Chimeric antigen receptor and methods of use thereof

ABSTRACT

The present disclosure provides a heterodimeric, conditionally active chimeric antigen receptor (CAR), and a nucleic acid comprising a nucleotide sequence encoding the CAR. The present disclosure provides cells genetically modified to produce the CAR. A CAR of the present disclosure can be used in various methods, which are also provided.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/765,585, filed Feb. 15, 2013, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.EY016546 and GM101782 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file, “UCSF-464WOSeqList_ST25.txt” created on Feb. 13, 2014 and having a size of 153 KB.The contents of the text file are incorporated by reference herein intheir entirety.

INTRODUCTION

In cell-based adoptive immunotherapy, immune cells isolated from apatient can be modified to express synthetic proteins that enable thecells to perform new therapeutic functions after they are subsequentlytransferred back into the patient. An example of such a syntheticprotein is a chimeric antigen receptor (CAR). An example of a currentlyused CAR is a fusion of an extracellular recognition domain (e.g., anantigen-binding domain), a transmembrane domain, and one or moreintracellular signaling domains. Upon antigen engagement, theintracellular signaling portion of the CAR can initiate anactivation-related response in an immune cell, such are release ofcytolytic molecules to induce tumor cell death, etc. However, such CARsare not capable of being pharmacologically controlled. There is a needin the art for a conditionally activatable CAR that can be controlledpharmacologically.

SUMMARY

The present disclosure provides a heterodimeric, conditionally activechimeric antigen receptor (CAR), and a nucleic acid comprising anucleotide sequence encoding the CAR. The present disclosure providescells genetically modified to produce the CAR. A CAR of the presentdisclosure can be used in various methods, which are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B provide nucleotide and amino acid sequences of thedomains of construct #122.

FIGS. 2A and 2B provide nucleotide and amino acid sequences of thedomains of construct #123.

FIGS. 3A and 3B provide nucleotide and amino acid sequences of thedomains of construct #125.

FIG. 4 provides nucleotide and amino acid sequences of the domains ofconstruct #126.

FIGS. 5A and 5B provide nucleotide and amino acid sequences of thedomains of construct #168.

FIGS. 6A-C provide nucleotide and amino acid sequences of the domains ofconstruct #169.

FIGS. 7A and 7B provide nucleotide and amino acid sequences of thedomains of construct #170.

FIGS. 8A and 8B provide nucleotide and amino acid sequences of thedomains of construct #197.

FIGS. 9A-C provide nucleotide and amino acid sequences of the domains ofconstruct #206.

FIGS. 10A and 10B provide nucleotide and amino acid sequences of thedomains of construct #207.

FIGS. 11A-C provide nucleotide and amino acid sequences of the domainsof construct #199.

FIG. 12 depicts IL-2 production triggered by five On-switch CARvariants.

FIG. 13 depicts IL-2 production by control Jurkat lines.

FIG. 14 depicts a comparison between CAR constructs “122+206” and“197+206”.

FIG. 15 depicts cytotoxicity data with the On-switch CAR “197+206.”

FIG. 16 depicts T cell activation data using CAR constructs “122+199”;“197+199”; and “122+168.”

FIG. 17 is a schematic representation of an exemplary On-switch CAR.

FIGS. 18A and 18B depict various exemplary On-switch CAR.

FIGS. 19A-G depict IL-2 production triggered by 3 different On-switchCAR variants recognizing human mesothelin.

FIGS. 20A-C depict IL-2 production triggered by an On-switch CAR variantwith a gibberellic acid responsive dimerization pair.

FIGS. 21A-D depict exemplary On-switch CARs and conventional CARs withvarious co-stimulatory domains.

FIGS. 22A and 22B provide nucleotide and amino acid sequences of thedomains of construct #270.

FIGS. 23A and 23B provide nucleotide and amino acid sequences of thedomains of construct #300.

FIGS. 24A and 24B provide nucleotide and amino acid sequences of thedomains of construct #336.

FIGS. 25A and 25B provide nucleotide and amino acid sequences of thedomains of construct #337.

FIGS. 26A and 26B provide nucleotide and amino acid sequences of thedomains of construct #357.

FIGS. 27A and 27B provide nucleotide and amino acid sequences of thedomains of construct #365.

FIGS. 28A and 28B provide nucleotide and amino acid sequences of thedomains of construct #366.

FIGS. 29A and 29B provide nucleotide and amino acid sequences of thedomains of construct #367.

FIGS. 30A and 30B provide nucleotide and amino acid sequences of thedomains of construct #398.

FIGS. 31A and 31B provide nucleotide and amino acid sequences of thedomains of construct #399.

FIGS. 32A and 32B provide nucleotide and amino acid sequences of thedomains of construct #400.

FIGS. 33A and 33B provide nucleotide and amino acid sequences of thedomains of construct #358.

DEFINITIONS

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-, double-, or multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein.

“Antibody fragments” comprise a portion of an intact antibody, forexample, the antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 (1995)); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, a designation reflecting the abilityto crystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. In some embodiments, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains,which enables the sFv to form the desired structure for antigen binding.For a review of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

As used herein, the term “affinity” refers to the equilibrium constantfor the reversible binding of two agents and is expressed as adissociation constant (Kd). Affinity can be at least 1-fold greater, atleast 2-fold greater, at least 3-fold greater, at least 4-fold greater,at least 5-fold greater, at least 6-fold greater, at least 7-foldgreater, at least 8-fold greater, at least 9-fold greater, at least10-fold greater, at least 20-fold greater, at least 30-fold greater, atleast 40-fold greater, at least 50-fold greater, at least 60-foldgreater, at least 70-fold greater, at least 80-fold greater, at least90-fold greater, at least 100-fold greater, or at least 1000-foldgreater, or more, than the affinity of an antibody for unrelated aminoacid sequences. Affinity of an antibody to a target protein can be, forexample, from about 100 nanomolar (nM) to about 0.1 nM, from about 100nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar(fM) or more. As used herein, the term “avidity” refers to theresistance of a complex of two or more agents to dissociation afterdilution. The terms “immunoreactive” and “preferentially binds” are usedinterchangeably herein with respect to antibodies and/or antigen-bindingfragments.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions, including interactions such as saltbridges and water bridges. Non-specific binding would refer to bindingwith an affinity of less than about 10⁻⁷ M, e.g., binding with anaffinity of 10⁻⁶ M, 10⁻⁵ M, 10⁻⁴ M, etc.

As used herein, the term “hinge region” refers to a flexible polypeptideconnector region (also referred to herein as “hinge” or “spacer”)providing structural flexibility and spacing to flanking polypeptideregions and can consist of natural or synthetic polypeptides. A “hingeregion” derived from an immunoglobulin (e.g., IgG1) is generally definedas stretching from Glu₂₁₆ to Pro₂₃₀ of human IgG1 (Burton (1985) Molec.Immunol., 22:161-206). Hinge regions of other IgG isotypes may bealigned with the IgG1 sequence by placing the first and last cysteineresidues forming inter-heavy chain disulfide (S—S) bonds in the samepositions. The hinge region may be of natural occurrence or non-naturaloccurrence, including but not limited to an altered hinge region asdescribed in U.S. Pat. No. 5,677,425. The hinge region can includecomplete hinge region derived from an antibody of a different class orsubclass from that of the CH1 domain. The term “hinge region” can alsoinclude regions derived from CD8 and other receptors that provide asimilar function in providing flexibility and spacing to flankingregions.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, the polypeptide will bepurified (1) to greater than 90%, greater than 95%, or greater than 98%,by weight of antibody as determined by the Lowry method, for example,more than 99% by weight, (2) to a degree sufficient to obtain at least15 residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (3) to homogeneity by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing ornonreducing conditions using Coomassie blue or silver stain. Isolatedpolypeptide includes the polypeptide in situ within recombinant cellssince at least one component of the polypeptide's natural environmentwill not be present. In some instances, isolated polypeptide will beprepared by at least one purification step.

As used herein, the term “immune cells” generally includes white bloodcells (leukocytes) which are derived from hematopoietic stem cells (HSC)produced in the bone marrow. “Immune cells” includes, e.g., lymphocytes(T cells, B cells, natural killer (NK) cells) and myeloid-derived cells(neutrophil, eosinophil, basophil, monocyte, macrophage, dendriticcells).

“T cell” includes all types of immune cells expressing CD3 includingT-helper cells (CD4⁺ cells), cytotoxic T-cells (CD8⁺ cells),T-regulatory cells (Treg) and gamma-delta T cells.

A “cytotoxic cell” includes CD8⁺ T cells, natural-killer (NK) cells, andneutrophils, which cells are capable of mediating cytotoxicityresponses.

As used herein, the term “stem cell” generally includes pluripotent ormultipotent stem cells. “Stem cells” includes, e.g., embryonic stemcells (ES); mesenchymal stem cells (MSC); induced-pluripotent stem cells(iPS); and committed progenitor cells (hematopoeitic stem cells (HSC);bone marrow derived cells, etc.).

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, e.g., in a human, and includes: (a)preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;(b) inhibiting the disease, i.e., arresting its development; and (c)relieving the disease, i.e., causing regression of the disease.

The terms “individual,” “subject,” “host,” and “patient,” usedinterchangeably herein, refer to a mammal, including, but not limitedto, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-humanprimates, humans, canines, felines, ungulates (e.g., equines, bovines,ovines, porcines, caprines), etc.

A “therapeutically effective amount” or “efficacious amount” refers tothe amount of an agent, or combined amounts of two agents, that, whenadministered to a mammal or other subject for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the agent(s),the disease and its severity and the age, weight, etc., of the subjectto be treated.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “achimeric antigen receptor” includes a plurality of such chimeric antigenreceptor and reference to “the dimerizer-binding pair” includesreference to one or more dimerizer-binding pairs and equivalents thereofknown to those skilled in the art, and so forth. It is further notedthat the claims may be drafted to exclude any optional element. As such,this statement is intended to serve as antecedent basis for use of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides a heterodimeric, conditionally activechimeric antigen receptor (CAR), and a nucleic acid comprising anucleotide sequence encoding the CAR. The present disclosure providescells genetically modified to produce the CAR. A CAR of the presentdisclosure can be used in various methods, which are also provided.

Heterodimeric, Conditionally Active Chimeric Antigen Receptor.

The present disclosure provides a heterodimeric, conditionally activechimeric antigen receptor, which, for simplicity, is referred to hereinas “CAR.”

In some embodiments, a CAR of the present disclosure comprises: a) afirst polypeptide comprising: i) a member of a specific binding pair(e.g., an antigen-binding domain); ii) a first modulatory domain; iii) afirst member of a dimerization pair; and iv) a transmembrane domaininterposed between the member of a specific binding pair (e.g., anantigen-binding domain) and the first modulatory domain; and b) a secondpolypeptide comprising: i) a transmembrane domain; ii) a secondmodulatory domain; iii) a second member of the dimerization pair; andiv) an intracellular signaling domain. The modulatory domain can be aco-stimulatory domain.

In some embodiments, a CAR of the present disclosure comprises: a) afirst polypeptide comprising: i) a member of a specific binding pair(e.g., an antigen-binding domain); ii) a first co-stimulatory domain;iii) a first member of a dimerization pair (e.g., a dimerizer-bindingpair); and iv) a transmembrane domain interposed between the member of aspecific binding pair (e.g., an antigen-binding domain) and the firstco-stimulatory domain; and b) a second polypeptide comprising: i) atransmembrane domain; ii) a second co-stimulatory domain; iii) a secondmember of the dimerization pair (e.g., the dimerizer-binding pair); andiv) an intracellular signaling domain.

In some embodiments, a CAR of the present disclosure comprises: a) afirst polypeptide comprising: i) a member of a specific binding pair(e.g., an antigen-binding domain); ii) a modulatory domain; iii) a firstmember of a dimerization pair (e.g., a dimerizer-binding pair); iv) atransmembrane domain interposed between the member of a specific bindingpair (e.g., an antigen-binding domain) and the modulatory domain; and b)a second polypeptide comprising: i) a second member of the dimerizationpair (e.g., the dimerizer-binding pair); and ii) an intracellularsignaling domain. The modulatory domain can be a co-stimulatory domain.

In some embodiments, a CAR of the present disclosure comprises: a) afirst polypeptide comprising: i) a member of a specific binding pair(e.g., an antigen-binding domain); ii) a co-stimulatory domain; iii) afirst member of a dimerization pair (e.g., a dimerizer-binding pair);iv) a transmembrane domain interposed between the member of a specificbinding pair (e.g., an antigen-binding domain) and the co-stimulatorydomain; and b) a second polypeptide comprising: i) a second member ofthe dimerization pair (e.g., the dimerizer-binding pair); and ii) anintracellular signaling domain.

An example of a subject CAR is represented schematically in FIG. 17. ACAR of the present disclosure can be present in the plasma membrane of aeukaryotic cell, e.g., a mammalian cell, where suitable mammalian cellsinclude, but are not limited to, a cytotoxic cell, a T lymphocyte, astem cell, a progeny of a stem cell, a progenitor cell, a progeny of aprogenitor cell, and an NK cell. When present in the plasma membrane ofa eukaryotic cell, a CAR of the present disclosure is active in thepresence of: 1) a dimerizing agent binds to the first and second membersof the dimerizer-binding pair in the CAR, or otherwise inducesdimerization of the first and second members of the dimer; and 2) afactor that binds the member of a specific binding pair (e.g., anantigen-binding domain), e.g., an antigen that binds the antigen-bindingdomain of the CAR. The factor that binds the member of the specificbinding pair is a second member of the specific binding pair. The secondmember of the specific binding pair can be a soluble (e.g., not bound toa cell) factor; a factor present on the surface of a cell such as atarget cell; a factor presented on a solid surface; a factor present ina lipid bilayer; and the like. Where the member of a specific bindingpair is an antibody, and the second member of the specific binding pairis an antigen, the antigen can be a soluble (e.g., not bound to a cell)antigen; an antigen present on the surface of a cell such as a targetcell; an antigen presented on a solid surface; an antigen present in alipid bilayer; and the like.

In some instances, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by a secondmember of a specific binding pair that binds the member of thespecific-binding pair of the CAR (e.g., an antigen that binds theantigen-binding domain of the CAR) and a dimerizing agent, increasesexpression of at least one nucleic acid in the cell. For example, insome cases, a CAR of the present disclosure, when present in the plasmamembrane of a eukaryotic cell, and when activated by an antigen thatbinds the antigen-binding domain of the CAR and a dimerizing agent,increases expression of at least one nucleic acid in the cell by atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 40%, at least about 50%, atleast about 75%, at least about 2-fold, at least about 2.5-fold, atleast about 5-fold, at least about 10-fold, or more than 10-fold,compared with the level of transcription of the nucleic acid in theabsence of the antigen and/or the dimerizing agent.

As an example, the second polypeptide of a CAR of the present disclosurecan include an immunoreceptor tyrosine-based activation motif(ITAM)-containing intracellular signaling polypeptide; in such cases, aCAR of the present disclosure, when present in the plasma membrane of aeukaryotic cell, and when activated by an antigen that binds theantigen-binding domain of the CAR and a dimerizing agent, increasesnuclear factor of activated T cells (NFAT)-dependent transcription.NFAT-dependent transcription includes transcription induced by anymember of the NFAT family, including, e.g., NFATc1, NFATc2, NFATc3,NFATc4, NFAT5; AP-1; Sp1; NKκB; and the like.

A CAR of the present disclosure, when present in the plasma membrane ofa eukaryotic cell, and when activated by an antigen that binds theantigen-binding domain of the CAR and a dimerizing agent, can, in someinstances, result in increased production of one or more cytokines bythe cell. For example, a CAR of the present disclosure, when present inthe plasma membrane of a eukaryotic cell, and when activated by anantigen that binds the antigen-binding domain of the CAR and adimerizing agent, can increase production of a cytokine by the cell byat least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 40%, at least about 50%,at least about 75%, at least about 2-fold, at least about 2.5-fold, atleast about 5-fold, at least about 10-fold, or more than 10-fold,compared with the amount of cytokine produced by the cell in the absenceof the antigen and/or the dimerizing agent. Cytokines whose productioncan be increased include, but are not limited to, an interferon, e.g.,IL-2, interferon gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α),IL-15, IL-12, IL-4, IL-5, IL-10; a chemokine; a growth factor; and thelike.

In some cases, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by an antigenthat binds the antigen-binding domain of the CAR and a dimerizing agent,can result in both an increase in transcription of a nucleic acid in thecell and an increase in production of a cytokine by the cell.

In some instances, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by a dimerizingagent, results in cytotoxic activity by the cell toward a target cellthat expresses on its cell surface an antigen to which theantigen-binding domain of the first polypeptide of the CAR binds. Forexample, where the eukaryotic cell is a cytotoxic cell (e.g., an NK cellor a cytotoxic T lymphocyte), a CAR of the present disclosure, whenpresent in the plasma membrane of the cell, and when activated by adimerizing agent, increases cytotoxic activity of the cell toward atarget cell that expresses on its cell surface an antigen to which theantigen-binding domain of the first polypeptide of the CAR binds. Forexample, where the eukaryotic cell is an NK cell or a T lymphocyte, aCAR of the present disclosure, when present in the plasma membrane ofthe cell, and when activated by a dimerizing agent, increases cytotoxicactivity of the cell by at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 40%,at least about 50%, at least about 75%, at least about 2-fold, at leastabout 2.5-fold, at least about 5-fold, at least about 10-fold, or morethan 10-fold, compared to the cytotoxic activity of the cell in theabsence of the dimerizing agent.

In some cases, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by an antigenthat binds the antigen-binding domain of the CAR and a dimerizing agent,can result in other CAR activation related events such as proliferationand expansion (either due to increased cellular division oranti-apoptotic responses).

In some cases, a CAR of the present disclosure, when present in theplasma membrane of a eukaryotic cell, and when activated by an antigenthat binds the antigen-binding domain of the CAR and a dimerizing agent,can result in other CAR activation related events such as intracellularsignaling modulation, cellular differentiation, or cell death.

A CAR of the present disclosure can be present in a eukaryotic cellmembrane, where the first and second polypeptides of the CAR are notcovalently linked to one another. A CAR of the present disclosure can bepresent in a eukaryotic cell membrane as a single heterodimer that isnot covalently linked to any other polypeptide in the membrane.Alternatively, a first CAR of the present disclosure can be present in aeukaryotic cell membrane as a heterodimer that is covalently ornon-covalently linked to a second CAR of the present disclosure. In somecases, the first and the second CAR are covalently linked via adisulfide bond formed between cysteines present in a hinge regionpresent in both the first polypeptide of the first CAR and the firstpolypeptide of the second CAR.

In some cases, a CAR of the present disclosure can be present in aeukaryotic cell membrane, where the first polypeptides of the CARcomprise an antibody fragment and the second polypeptides of the CARcomprise a signal transducing domain derived from a cytokine receptor,such that, upon dimerization, the CAR may represent aheterodimeric-signalobody CAR, e.g., a signalobody composed of at leasttwo independent polypeptides. A “signalobody”, as it is known in theart, is a single chimeric macromolecule composed of an antibody fragmentand a signal transduction domain derived from a cytokine receptor. Incertain instances, a heterodimeric-signalobody CAR of the presentdisclosure, when present in the cell membrane of a eukaryotic cell,dimerized by a dimerizer, and activated by an antigen, e.g., anoligomerized antigen, may induce the oligomerization of theheterodimeric-signalobody CAR. Such ligand-induced oligomerization of aheterodimeric-signalobody CAR may activate, e.g., increase, orperpetuate, e.g., maintain, signal transduction, e.g., ligand-inducedoligomerization of a heterodimeric-signalobody CAR may transmit a signaleliciting a cellular response. In some instances, a plurality ofheterodimeric-signalobody CARs may be utilized combinatorially to elicita desired cellular response.

Member of a Specific Binding Pair

A CAR of the present disclosure includes a member of a specific bindingpair. Specific binding pairs include, but are not limited to,antigen-antibody binding pairs; ligand-receptor binding pairs; and thelike. Thus, a member of a specific binding pair suitable for use in aCAR of the present disclosure includes an antigen; an antibody; aligand; and a ligand-binding receptor.

Antigen-Binding Domain

An antigen-binding domain suitable for use in a CAR of the presentdisclosure can be any antigen-binding polypeptide, a wide variety ofwhich are known in the art. In some instances, the antigen-bindingdomain is a single chain Fv (scFv). Other antibody based recognitiondomains (cAb VHH (camelid antibody variable domains) and humanizedversions, IgNAR VH (shark antibody variable domains) and humanizedversions, sdAb VH (single domain antibody variable domains) and“camelized” antibody variable domains are suitable for use. In someinstances, T-cell receptor (TCR) based recognition domains such assingle chain TCR (scTv, single chain two-domain TCR containing VαVβ) arealso suitable for use.

An antigen-binding domain suitable for use in a CAR of the presentdisclosure can have a variety of antigen-binding specificities. In somecases, the antigen-binding domain is specific for an epitope present inan antigen that is expressed by (synthesized by) a cancer cell, i.e., acancer cell associated antigen. The cancer cell associated antigen canbe an antigen associated with, e.g., a breast cancer cell, a B celllymphoma, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostatecancer cell, a mesothelioma, a lung cancer cell (e.g., a small cell lungcancer cell), a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovariancancer cell, a prostate cancer cell, a mesothelioma cell, a lung cancercell (e.g., a small cell lung cancer cell), a melanoma cell, a chroniclymphocytic leukemia cell, an acute lymphocytic leukemia cell, aneuroblastoma cell, a glioma, a glioblastoma, a medulloblastoma, acolorectal cancer cell, etc. A cancer cell associated antigen may alsobe expressed by a non-cancerous cell.

Non-limiting examples of antigens to which an antigen-binding domain ofa subject CAR can bind include, e.g., CD19, CD20, CD38, CD30, Her2/neu,ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA),epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelialgrowth factor receptor-2 (VEGFR2), high molecular weight-melanomaassociated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like.

Ligand

In some cases, a member of a specific binding pair suitable for use in asubject CAR is a ligand for a receptor. Ligands include, but are notlimited to, cytokines (e.g., IL-13, etc.); growth factors (e.g.,heregulin; vascular endothelial growth factor (VEGF); and the like); anintegrin-binding peptide (e.g., a peptide comprising the sequenceArg-Gly-Asp); and the like.

Where the member of a specific binding pair in a subject CAR is aligand, the CAR can be activated in the presence of both a dimerizeragent and a second member of the specific binding pair, where the secondmember of the specific binding pair is a receptor for the ligand. Forexample, where the ligand is VEGF, the second member of the specificbinding pair can be a VEGF receptor, including a soluble VEGF receptor.As another example, where the ligand is heregulin, the second member ofthe specific binding pair can be Her2.

Receptors

As noted above, in some cases, the member of a specific binding pairthat is included in a subject CAR is a receptor, e.g., a receptor for aligand, a co-receptor, etc. The receptor can be a ligand-bindingfragment of a receptor. Suitable receptors include, but are not limitedto, a growth factor receptor (e.g., a VEGF receptor); a killer celllectin-like receptor subfamily K, member 1 (NKG2D) polypeptide (receptorfor MICA, MICB, and ULB6); a cytokine receptor (e.g., an IL-13 receptor;an IL-2 receptor; etc.); Her2; CD27; a natural cytotoxicity receptor(NCR) (e.g., NKP30 (NCR3/CD337) polypeptide (receptor forHLA-B-associated transcript 3 (BAT3) and B7-H6); etc.); etc.

Hinge Region

In some cases, the first polypeptide of a subject CAR comprises a hingeregion (also referred to herein as a “spacer”), where the hinge regionis interposed between the antigen-binding domain and the transmembranedomain. In some cases, the hinge region is an immunoglobulin heavy chainhinge region. In some cases, the hinge region is a hinge regionpolypeptide derived from a receptor (e.g., a CD8-derived hinge region).

The hinge region can have a length of from about 4 amino acids to about50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aato about 15 aa, from about 15 aa to about 20 aa, from about 20 aa toabout 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about40 aa, or from about 40 aa to about 50 aa.

Suitable spacers can be readily selected and can be of any of a numberof suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary spacers include glycine polymers (G)_(n), glycine-serinepolymers (including, for example, (GS)_(n), (GSGGS)_(n) (SEQ ID NO:37)and (GGGS)_(n) (SEQ ID NO:38), where n is an integer of at least one),glycine-alanine polymers, alanine-serine polymers, and other flexiblelinkers known in the art. Glycine and glycine-serine polymers can beused; both Gly and Ser are relatively unstructured, and therefore canserve as a neutral tether between components. Glycine polymers can beused; glycine accesses significantly more phi-psi space than evenalanine, and is much less restricted than residues with longer sidechains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).Exemplary spacers can comprise amino acid sequences including, but notlimited to, GGSG (SEQ ID NO:39), GGSGG (SEQ ID NO:40), GSGSG (SEQ IDNO:41), GSGGG (SEQ ID NO:42), GGGSG (SEQ ID NO:43), GSSSG (SEQ IDNO:44), and the like.

In some cases, the hinge region in the first polypeptide of a subjectCAR includes at least one cysteine. For example, in some cases, thehinge region can include the sequence Cys-Pro-Pro-Cys. If present, acysteine in the hinge region of a first CAR can be available to form adisulfide bond with a hinge region in a second CAR.

Immunoglobulin hinge region amino acid sequences are known in the art;see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Hucket al. (1986) Nucl. Acids Res. 14:1779. As non-limiting examples, animmunoglobulin hinge region can include one of the following amino acidsequences: DKTHT (SEQ ID NO:45); CPPC (SEQ ID NO:46); CPEPKSCDTPPPCPR(SEQ ID NO:47) (see, e.g., Glaser et al. (2005) J. Biol. Chem.280:41494); ELKTPLGDTTHT (SEQ ID NO:48); KSCDKTHTCP (SEQ ID NO:49);KCCVDCP (SEQ ID NO:50); KYGPPCP (SEQ ID NO:51); EPKSCDKTHTCPPCP (SEQ IDNO:52) (human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO:53) (human IgG2hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO:54) (human IgG3 hinge);SPNMVPHAHHAQ (SEQ ID NO:55) (human IgG4 hinge); and the like.

The hinge region can comprise an amino acid sequence of a human IgG1,IgG2, IgG3, or IgG4, hinge region. The hinge region can include one ormore amino acid substitutions and/or insertions and/or deletionscompared to a wild-type (naturally-occurring) hinge region. For example,His₂₂₉ of human IgG1 hinge can be substituted with Tyr, so that thehinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO:52); see,e.g., Yan et al. (2012) J. Biol. Chem. 287:5891.

The hinge region can comprise an amino acid sequence derived from humanCD8; e.g., the hinge region can comprise the amino acid sequence:

(SEQ ID NO: 56) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD,or a variant thereof.

Transmembrane Domain

The first and the second polypeptides of a CAR of the present disclosureinclude transmembrane domains for insertion into a eukaryotic cellmembrane. The transmembrane domain of the first polypeptide isinterposed between the antigen-binding domain and the co-stimulatorydomain. Where the first polypeptide includes a hinge region, thetransmembrane domain is interposed between the hinge region and theco-stimulatory domain, such that the first polypeptide comprises, inorder from the amino terminus (N-terminus) to the carboxyl terminus(C-terminus): an antigen-binding domain; a hinge region; a transmembranedomain; a first co-stimulatory domain; and a first member of adimerizer-binding pair.

The transmembrane domain of the second polypeptide is at or near theN-terminus of the polypeptide, such that the second polypeptidecomprises, in order from N-terminus to C-terminus: a transmembranedomain; a second co-stimulatory domain; a second member of thedimerizer-binding pair; and an intracellular signaling domain.

Any transmembrane (TM) domain that provides for insertion of apolypeptide into the cell membrane of a eukaryotic (e.g., mammalian)cell is suitable for use. As one non-limiting example, the TM sequenceIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:30) can be used. Additionalnon-limiting examples of suitable TM sequences include: a) CD8 betaderived: LGLLVAGVLVLLVSLGVAIHLCC (SEQ ID NO:57); b) CD4 derived:ALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO:58); c) CD3 zeta derived:LCYLLDGILFIYGVILTALFLRV (SEQ ID NO:59); d) CD28 derived:WVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO:60); e) CD134 (OX40) derived:VAAILGLGLVLGLLGPLAILLALYLL (SEQ ID NO:61); and f) CD7 derived:ALPAALAVISFLLGLGLGVACVLA (SEQ ID NO:62).

Linkers

In some cases, a first polypeptide of a subject CAR includes a linkerbetween any two adjacent domains. For example, a linker can be disposedbetween the transmembrane domain and the first co-stimulatory domain ofthe first polypeptide. As another example, a linker can be disposedbetween the first co-stimulatory domain and the first member of adimerizer-binding pair of the first polypeptide. As another example, alinker can be disposed between the transmembrane domain and the secondco-stimulatory domain of the second polypeptide. As another example, alinker can be disposed between the second co-stimulatory domain and thesecond member of the dimerizer-binding pair of the second polypeptide.As another example, a linker can be disposed between the second memberof the dimerizer-binding pair and the intracellular signaling domain ofthe second polypeptide.

The linker peptide may have any of a variety of amino acid sequences.Proteins can be joined by a spacer peptide, generally of a flexiblenature, although other chemical linkages are not excluded. A linker canbe a peptide of between about 6 and about 40 amino acids in length, orbetween about 6 and about 25 amino acids in length. These linkers can beproduced by using synthetic, linker-encoding oligonucleotides to couplethe proteins. Peptide linkers with a degree of flexibility can be used.The linking peptides may have virtually any amino acid sequence, bearingin mind that suitable linkers will have a sequence that results in agenerally flexible peptide. The use of small amino acids, such asglycine and alanine, are of use in creating a flexible peptide. Thecreation of such sequences is routine to those of skill in the art.

Suitable linkers can be readily selected and can be of any of a suitableof different lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), GSGGS_(n)(SEQ ID NO:37) and GGGS_(n) (SEQ ID NO:38), where n is an integer of atleast one), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers known in the art. Glycine and glycine-serine polymersare of interest since both of these amino acids are relativelyunstructured, and therefore may serve as a neutral tether betweencomponents. Glycine polymers are of particular interest since glycineaccesses significantly more phi-psi space than even alanine, and is muchless restricted than residues with longer side chains (see Scheraga,Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkersinclude, but are not limited GGSG (SEQ ID NO:39), GGSGG (SEQ ID NO:40),GSGSG (SEQ ID NO:41), GSGGG (SEQ ID NO:42), GGGSG (SEQ ID NO:43), GSSSG(SEQ ID NO:44), and the like. The ordinarily skilled artisan willrecognize that design of a peptide conjugated to any elements describedabove can include linkers that are all or partially flexible, such thatthe linker can include a flexible linker as well as one or more portionsthat confer less flexible structure.

Modulatory Domains

Modulatory domains suitable for use in a CAR of the present disclosureinclude co-stimulatory domains.

In some cases, the modulatory domain on the first polypeptide of asubject CAR has substantially the same amino acid sequence as themodulatory domain on the second polypeptide of the CAR. For example, insome cases, the modulatory domain on the first polypeptide of a CARcomprises an amino acid sequence that is at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100%, identical tothe amino acid sequence of the modulatory domain on the secondpolypeptide of the CAR. The modulatory domain of the first polypeptideof a subject CAR can have substantially the same length as themodulatory domain of the second polypeptide of a subject CAR; e.g., thefirst and second modulatory domains can differ in length from oneanother by fewer than 10 amino acids, or fewer than 5 amino acids. Insome cases, the first and second modulatory domains have the samelength.

A modulatory domain suitable for inclusion in the first and the secondpolypeptide of a subject CAR can have a length of from about 30 aminoacids to about 70 amino acids (aa), e.g., a modulatory domain can have alength of from about 30 aa to about 35 aa, from about 35 aa to about 40aa, from about 40 aa to about 45 aa, from about 45 aa to about 50 aa,from about 50 aa to about 55 aa, from about 55 aa to about 60 aa, fromabout 60 aa to about 65 aa, or from about 65 aa to about 70 aa. In othercases, modulatory domain can have a length of from about 70 aa to about100 aa, from about 100 aa to about 200 aa, or greater than 200 aa.

Co-stimulatory domains suitable for use in a CAR of the presentdisclosure are generally polypeptides derived from receptors. In someembodiments, co-stimulatory domains homodimerize. A subjectco-stimulatory domain can be an intracellular portion of a transmembraneprotein (i.e., the co-stimulatory domain can be derived from atransmembrane protein). Non-limiting examples of suitable co-stimulatorypolypeptides include, but are not limited to, 4-1BB (CD137), CD28, ICOS,OX-40, BTLA, CD27, CD30, GITR, and HVEM.

In some cases, the co-stimulatory domain on the first polypeptide of asubject CAR has substantially the same amino acid sequence as theco-stimulatory domain on the second polypeptide of the CAR. For example,in some cases, the co-stimulatory domain on the first polypeptide of aCAR comprises an amino acid sequence that is at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%,identical to the amino acid sequence of the co-stimulatory domain on thesecond polypeptide of the CAR. The co-stimulatory domain of the firstpolypeptide of a subject CAR can have substantially the same length asthe co-stimulatory domain of the second polypeptide of a subject CAR;e.g., the first and second co-stimulatory domains can differ in lengthfrom one another by fewer than 10 amino acids, or fewer than 5 aminoacids. In some cases, the first and second co-stimulatory domains havethe same length.

A co-stimulatory domain suitable for inclusion in the first and thesecond polypeptide of a subject CAR can have a length of from about 30amino acids to about 70 amino acids (aa), e.g., a co-stimulatory domaincan have a length of from about 30 aa to about 35 aa, from about 35 aato about 40 aa, from about 40 aa to about 45 aa, from about 45 aa toabout 50 aa, from about 50 aa to about 55 aa, from about 55 aa to about60 aa, from about 60 aa to about 65 aa, or from about 65 aa to about 70aa. In other cases, the co-stimulatory domain can have a length of fromabout 70 aa to about 100 aa, from about 100 aa to about 200 aa, orgreater than 200 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein 4-1BB (also known asTNFRSF9; CD137; 4-1BB; CDw137; ILA; etc.). For example, a suitableco-stimulatory domain can comprise an amino acid sequence having atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, or 100% amino acid sequenceidentity to the following amino acid sequence:KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:24). In some ofthese embodiments, the co-stimulatory domain of both the first and thesecond polypeptide has a length of from about 30 aa to about 35 aa, fromabout 35 aa to about 40 aa, from about 40 aa to about 45 aa, from about45 aa to about 50 aa, from about 50 aa to about 55 aa, from about 55 aato about 60 aa, from about 60 aa to about 65 aa, or from about 65 aa toabout 70 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD28 (also known asTp44). For example, a suitable co-stimulatory domain can comprise anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, or 100% amino acid sequence identity to the following amino acidsequence:

(SEQ ID NO: 63) FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.In some of these embodiments, the co-stimulatory domain of both thefirst and the second polypeptide has a length of from about 30 aa toabout 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa,from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, orfrom about 65 aa to about 70 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein ICOS (also known asAILIM, CD278, and CVID1). For example, a suitable co-stimulatory domaincan comprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100% amino acid sequence identity to thefollowing amino acid sequence: TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQID NO:64). In some of these embodiments, the co-stimulatory domain ofboth the first and the second polypeptide has a length of from about 30aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa toabout 45 aa, from about 45 aa to about 50 aa, from about 50 aa to about55 aa, from about 55 aa to about 60 aa, from about 60 aa to about 65 aa,or from about 65 aa to about 70 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein OX-40 (also known asTNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). For example, asuitable co-stimulatory domain can comprise an amino acid sequencehaving at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or 100% aminoacid sequence identity to the following amino acid sequence:RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO:65). In some of theseembodiments, the co-stimulatory domain of both the first and the secondpolypeptide has a length of from about 30 aa to about 35 aa, from about35 aa to about 40 aa, from about 40 aa to about 45 aa, from about 45 aato about 50 aa, from about 50 aa to about 55 aa, from about 55 aa toabout 60 aa, from about 60 aa to about 65 aa, or from about 65 aa toabout 70 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein BTLA (also known asBTLA1 and CD272). For example, a suitable co-stimulatory domain cancomprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100% amino acid sequence identity to thefollowing amino acid sequence:

(SEQ ID NO: 66) CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLAR NVKEAPTEYASICVRS.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD27 (also known asS152, T14, TNFRSF7, and Tp55). For example, a suitable co-stimulatorydomain can comprise an amino acid sequence having at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, or 100% amino acid sequence identity to thefollowing amino acid sequence:HQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:67). Insome of these embodiments, the co-stimulatory domain of both the firstand the second polypeptide has a length of from about 30 aa to about 35aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa,from about 45 aa to about 50 aa, from about 50 aa to about 55 aa, fromabout 55 aa to about 60 aa, from about 60 aa to about 65 aa, or fromabout 65 aa to about 70 aa.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein CD30 (also known asTNFRSF8, D1S166E, and Ki-1). For example, a suitable co-stimulatorydomain can comprise an amino acid sequence having at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 98%, or 100% amino acid sequence identity to acontiguous stretch of from about 100 amino acids to about 110 aminoacids (aa), from about 110 aa to about 115 aa, from about 115 aa toabout 120 aa, from about 120 aa to about 130 aa, from about 130 aa toabout 140 aa, from about 140 aa to about 150 aa, from about 150 aa toabout 160 aa, or from about 160 aa to about 185 aa of the followingamino acid sequence:

(SEQ ID NO: 68) RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK.

In some cases, the co-stimulatory domain is derived from anintracellular portion of the transmembrane protein GITR (also known asTNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitableco-stimulatory domain can comprise an amino acid sequence having atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, or 100% amino acid sequenceidentity to the following amino acid sequence:HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV (SEQ ID NO:69).In some of these embodiments, the co-stimulatory domain of both thefirst and the second polypeptide has a length of from about 30 aa toabout 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa,from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, orfrom about 65 aa to about 70 aa.

In some cases, the co-stimulatory domain derived from an intracellularportion of the transmembrane protein HVEM (also known as TNFRSF14,RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, asuitable co-stimulatory domain can comprise an amino acid sequencehaving at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or 100% aminoacid sequence identity to the following amino acid sequence:CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRS PNH (SEQ IDNO:70). In some of these embodiments, the co-stimulatory domain of boththe first and the second polypeptide has a length of from about 30 aa toabout 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about45 aa, from about 45 aa to about 50 aa, from about 50 aa to about 55 aa,from about 55 aa to about 60 aa, from about 60 aa to about 65 aa, orfrom about 65 aa to about 70 aa.

Dimer Pairs

Dimer pairs suitable for use in a subject CAR include dimerizer-bindingpairs. Dimerizer-binding pairs suitable for use in a CAR of the presentdisclosure are in some embodiments polypeptides that bind to a differentsite of the same molecule (referred to herein as a “dimerizer”). In thepresence of a dimerizer, both members of the dimerizer-binding pair bindto a different site of the dimerizer and are thus brought into proximitywith one another. In some embodiments, binding to the dimerizer isreversible. In some embodiments, binding to the dimerizer isirreversible. In some embodiments, binding to the dimerizer isnon-covalent. In some embodiments, binding to the dimerizer is covalent.

Other dimer pairs suitable for use include dimerizer-binding pairs thatdimerize upon binding of a first member of a dimer pair to a dimerizingagent, where the dimerizing agent induces a conformational change in thefirst member of the dimer pair, and where the conformational changeallows the first member of the dimer pair to bind (covalently ornon-covalently) to a second member of the dimer pair.

Other dimer pairs suitable for use include dimer pairs in which exposureto light (e.g., blue light) induces dimerization of the dimer pair.

Regardless of the mechanism, the dimer pair will dimerize upon exposureto an agent that induces dimerization, where the agent is in some casesa small molecule, or, in other cases, light. Thus, for simplicity, thediscussion below referring to “dimerizer-binding pairs” includes dimerpairs that dimerize regardless of the mechanism.

Non-limiting examples of suitable dimers (e.g., dimerizer-binding pairs)include, but are not limited to:

-   a) FK506 binding protein (FKBP) and FKBP;-   b) FKBP and calcineurin catalytic subunit A (CnA);-   c) FKBP and cyclophilin;-   d) FKBP and FKBP-rapamycin associated protein (FRB);-   e) gyrase B (GyrB) and GyrB;-   f) dihydrofolate reductase (DHFR) and DHFR;-   g) DmrB and DmrB;-   h) PYL and ABI;-   i) Cry2 and CIB1; and-   j) GAI and GID1.

A first or a second member of a dimer (e.g., a dimerizer-binding pair)of a subject CAR can have a length of from about 50 amino acids to about300 amino acids or more; e.g., a first or a second member of a dimer(e.g., a dimerizer-binding pair) of a subject CAR can have a length offrom about 50 aa to about 100 aa, from about 100 aa to about 150 aa,from about 150 aa to about 200 aa, from about 200 aa to about 250 aa,from about 250 aa to about 300 aa, or more than 300 aa.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) of asubject CAR is derived from FKBP. For example, a suitabledimerizer-binding pair member can comprise an amino acid sequence havingat least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or 100% amino acidsequence identity to the following amino acid sequence:

(SEQ ID NO: 12) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLV FDVELLKLE.

In some cases, a member of a dimerizer-binding pair of a subject CAR isderived from calcineurin catalytic subunit A (also known as PPP3CA;CALN; CALNA; CALNA1; CCN1; CNA1; PPP2B; CAM-PRP catalytic subunit;calcineurin A alpha; calmodulin-dependent calcineurin A subunit alphaisoform; protein phosphatase 2B, catalytic subunit, alpha isoform;etc.). For example, a suitable dimerizer-binding pair member cancomprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100% amino acid sequence identity to thefollowing amino acid sequence (PP2Ac domain):

(SEQ ID NO: 71) LEESVALRIITEGASILRQEKNLLDIDAPVTVCGDIHGQFFDLMKLFEVGGSPANTRYLFLGDYVDRGYFSIECVLYLWALKILYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALMNQQFLCVHGGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLEDFGNEKTQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYRKSQTTGFPSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSPHPY WLPNFM.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from cyclophilin (also known cyclophilin A, PPIA, CYPA, CYPH,PPIase A, etc.). For example, a suitable dimerizer-binding pair membercan comprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100% amino acid sequence identity to thefollowing amino acid sequence:

(SEQ ID NO: 72) MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGFMCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLDGKHVVFGKVKEGMNIVEAMERFGSRNG KTSKKITIADCGQLE.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from MTOR (also known as FKBP-rapamycin associated protein;FK506 binding protein 12-rapamycin associated protein 1; FK506 bindingprotein 12-rapamycin associated protein 2; FK506-binding protein12-rapamycin complex-associated protein 1; FRAP; FRAP1; FRAP2; RAFT1;and RAPT1). For example, a suitable dimerizer-binding pair member cancomprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100% amino acid sequence identity to thefollowing amino acid sequence (also known as “Frb”: Fkbp-RapamycinBinding Domain):

(SEQ ID NO: 14) MILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from GyrB (also known as DNA gyrase subunit B). For example, asuitable dimerizer-binding pair member can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or 100%amino acid sequence identity to a contiguous stretch of from about 100amino acids to about 200 amino acids (aa), from about 200 aa to about300 aa, from about 300 aa to about 400 aa, from about 400 aa to about500 aa, from about 500 aa to about 600 aa, from about 600 aa to about700 aa, or from about 700 aa to about 800 aa, of the following GyrBamino acid sequence from Escherichia coli (or to the DNA gyrase subunitB sequence from any organism):

(SEQ ID NO: 73) MSNSYDSSSIKVLKGLDAVRKRPGMYIGDTDDGTGLHHMVFEVVDNAIDEALAGHCKEIIVTIHADNSVSVQDDGRGIPTGIHPEEGVSAAEVIMTVLHAGGKFDDNSYKVSGGLHGVGVSVVNALSQKLELVIQREGKIHRQIYEHGVPQAPLAVTGETEKTGTMVRFWPSLETFTNVTEFEYEILAKRLRELSFLNSGVSIRLRDKRDGKEDHFHYEGGIKAFVEYLNKNKTPIHPNIFYFSTEKDGIGVEVALQWNDGFQENIYCFTNNIPQRDGGTHLAGFRAAMTRTLNAYMDKEGYSKKAKVSATGDDAREGLIAVVSVKVPDPKFSSQTKDKLVSSEVKSAVEQQMNELLAEYLLENPTDAKIVVGKIIDAARAREAARRAREMTRRKGALDLAGLPGKLADCQERDPALSELYLVEGDSAGGSAKQGRNRKNQAILPLKGKILNVEKARFDKMLSSQEVATLITALGCGIGRDEYNPDKLRYHSIIIMTDADVDGSHIRTLLLTFFYRQMPEIVERGHVYIAQPPLYKVKKGKQEQYIKDDEAMDQYQISIALDGATLHTNASAPALAGEALEKLVSEYNATQKMINRMERRYPKAMLKELIYQPTLTEADLSDEQTVTRWVNALVSELNDKEQHGSQWKFDVHTNAEQNLFEPIVRVRTHGVDTDYPLDHEFITGGEYRRICTLGEKLRGLLEEDAFIERGERRQPVASFEQALDWLVKESRRGLSIQRYKGLGEMNPEQLWETTMDPESRRMLRVTVKDAIAADQLFTTLMGDAVEPRRAFIEENALKAA NIDI.In some cases, a member of a dimerizer-binding pair comprises an aminoacid sequence having at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,or 100% amino acid sequence identity to amino acids 1-220 of theabove-listed GyrB amino acid sequence from Escherichia coli.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from DHFR (also known as dihydrofolate reductase, DHFRP1, andDYR). For example, a suitable dimerizer-binding pair member can comprisean amino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, or 100% amino acid sequence identity to the following amino acidsequence:

(SEQ ID NO: 74) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from the DmrB binding domain (i.e., DmrB homodimerizationdomain). For example, a suitable dimerizer-binding pair member cancomprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100% amino acid sequence identity to thefollowing amino acid sequence:

(SEQ ID NO: 75) MASRGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHAT LVFDVELLKLE.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from a PYL protein (also known as abscisic acid receptor and asRCAR). For example a member of a subject dimerizer-binding pair can bederived from proteins such as those of Arabidopsis thaliana: PYR1,RCAR1(PYL9), PYL1, PYL2, PYL3, PYL4, PYL5, PYL6, PYL7, PYL8 (RCAR3),PYL10, PYL11, PYL12, PYL13. For example, a suitable dimerizer-bindingpair member can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100% amino acid sequenceidentity to any of the following amino acid sequences:

PYL10: (SEQ ID NO: 76)MNGDETKKVESEYIKKHHRHELVESQCSSTLVKHIKAPLHLVWSIVRRFDEPQKYKPFISRCVVQGKKLEVGSVREVDLKSGLPATKSTEVLEILDDNEHILGIRIVGGDHRLKNYSSTISLHSETIDGKTGTLAIESFVVDVPEGNTKEETCFFVEALIQCNLNSLADVTE RLQAESMEKKI.PYL11: (SEQ ID NO: 77)METSQKYHTCGSTLVQTIDAPLSLVWSILRRFDNPQAYKQFVKTCNLSSGDGGEGSVREVTVVSGLPAEFSRERLDELDDESHVMMISIIGGDHRLVNYRSKTMAFVAADTEEKTVVVESYVVDVPEGNSEEETTSFADTIVGFNLKSLAKLSERVAHLKL PYL12: (SEQ ID NO: 78)MKTSQEQHVCGSTVVQTINAPLPLVWSILRRFDNPKTFKHFVKTCKLRSGDGGEGSVREVTVVSDLPASFSLERLDELDDESHVMVISIIGGDHRLVNYQSKTTVFVAAEEEKTVVVESYVVDVPEGNTEEETTLFADTIVGCNLRSLAKLSEKMMELT. PYL13: (SEQ ID NO: 79)MESSKQKRCRSSVVETIEAPLPLVWSILRSFDKPQAYQRFVKSCTMRSGGGGGKGGEGKGSVRDVTLVSGFPADFSTERLEELDDESHVMVVSIIGGNHRLVNYKSKTKVVASPEDMAKKTVVVESYVVDVPEGTSEEDTIFFVDNIIRYNLTSLAKLTKKMMK. PYL1: (SEQ ID NO:80) MANSESSSSPVNEEENSQRISTLHHQTMPSDLTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMNRNNNNNNSSQVR. PYL2: (SEQ ID NO:81) MSSSPAVKGLTDEEQKTLEPVIKTYHQFEPDPTTCTSLITQRIHAPASVVWPLIRRFDNPERYKHFVKRCRLISGDGDVGSVREVTVISGLPASTSTERLEFVDDDHRVLSFRVVGGEHRLKNYKSVTSVNEFLNQDSGKVYTVVLESYTVDIPEGNTEEDTKMFVDTVVKLNLQKLGVAATSAPMHDDE. PYL3: (SEQ ID NO: 82)MNLAPIHDPSSSSTTTTSSSTPYGLTKDEFSTLDSIIRTHHTFPRSPNTCTSLIAHRVDAPAHAIWRFVRDFANPNKYKHFIKSCTIRVNGNGIKEIKVGTIREVSVVSGLPASTSVEILEVLDEEKRILSFRVLGGEHRLNNYRSVTSVNEFVVLEKDKKKRVYSVVLESYIVDIPQGNTEEDTRMFVDTVVKSNLQNLAVISTASPT. PYL4: (SEQ ID NO: 83)MLAVHRPSSAVSDGDSVQIPMMIASFQKRFPSLSRDSTAARFHTHEVGPNQCCSAVIQEISAPISTVWSVVRRFDNPQAYKHFLKSCSVIGGDGDNVGSLRQVHVVSGLPAASSTERLDILDDERHVISFSVVGGDHRLSNYRSVTTLHPSPISGTVVVESYVVDVPPGNTKEETCDFVDVIVRCNLQSLAKIAENTAAESKKKMSL. PYL5: (SEQ ID NO: 84)MRSPVQLQHGSDATNGFHTLQPHDQTDGPIKRVCLTRGMHVPEHVAMHHTHDVGPDQCCSSVVQMIHAPPESVWALVRRFDNPKVYKNFIRQCRIVQGDGLHVGDLREVMVVSGLPAVSSTERLEILDEERHVISFSVVGGDHRLKNYRSVTTLHASDDEGTVVVESYIVDVPPGNTEEETLSFVDTIVRCNLQSLARSTNRQ. PYL6: (SEQ ID NO: 85)MPTSIQFQRSSTAAEAANATVRNYPHHHQKQVQKVSLTRGMADVPEHVELSHTHVVGPSQCFSVVVQDVEAPVSTVWSILSRFEHPQAYKHFVKSCHVVIGDGREVGSVREVRVVSGLPAAFSLERLEIMDDDRHVISFSVVGGDHRLMNYKSVTTVHESEEDSDGKKRTRVVESYVVDVPAGNDKEETCSFADTIVRCNLQSLAKLAENTSKFS. PYL7: (SEQ ID NO: 86)MEMIGGDDTDTEMYGALVTAQSLRLRHLHHCRENQCTSVLVKYIQAPVHLVWSLVRRFDQPQKYKPFISRCTVNGDPEIGCLREVNVKSGLPATTSTERLEQLDDEEHILGINIIGGDHRLKNYSSILTVHPEMIDGRSGTMVMESFVVDVPQGNTKDDTCYFVESLIKCNLKSLACVSERLAAQDITNSIATFCNASNGYREKNHTETNL. PYL8: (SEQ ID NO: 87)MEANGIENLTNPNQEREFIRRHHKHELVDNQCSSTLVKHINAPVHIVWSLVRRFDQPQKYKPFISRCVVKGNMEIGTVREVDVKSGLPATRSTERLELLDDNEHILSIRIVGGDHRLKNYSSIISLHPETIEGRIGTLVIESFVVDVPEGNTKDETCYFVEALIKCNLKSLADISERLAVQDTTESRV. PYL9: (SEQ ID NO: 88)MMDGVEGGTAMYGGLETVQYVRTHHQHLCRENQCTSALVKHIKAPLHLVWSLVRRFDQPQKYKPFVSRCTVIGDPEIGSLREVNVKSGLPATTSTERLELLDDEEHILGIKIIGGDHRLKNYSSILTVHPEIIEGRAGTMVIESFVVDVPQGNTKDETCYFVEALIRCNLKSLADVSERLASQDITQ. PYR1: (SEQ ID NO: 89)MPSELTPEERSELKNSIAEFHTYQLDPGSCSSLHAQRIHAPPELVWSIVRRFDKPQTYKHFIKSCSVEQNFEMRVGCTRDVIVISGLPANTSTERLDILDDERRVTGFSIIGGEHRLTNYKSVTTVHRFEKENRIWTVVLESYVVDMPEGNSEDDTRMFADTVVKLNLQKLATVAEAMARNSGDGSGSQVT.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from an ABI protein (also known as Abscisic Acid-Insensitive).For example a member of a subject dimerizer-binding pair can be derivedfrom proteins such as those of Arabidopsis thaliana: ABI1 (Also known asABSCISIC ACID-INSENSITIVE 1, Protein phosphatase 2C 56, AtPP2C56, P2C56,and PP2C ABI1) and/or ABI2 (also known as P2C77, Protein phosphatase 2C77, AtPP2C77, ABSCISIC ACID-INSENSITIVE 2, Protein phosphatase 2C ABI2,and PP2C ABI2). For example, a suitable dimerizer-binding pair membercan comprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100% amino acid sequence identity to a contiguousstretch of from about 100 amino acids to about 110 amino acids (aa),from about 110 aa to about 115 aa, from about 115 aa to about 120 aa,from about 120 aa to about 130 aa, from about 130 aa to about 140 aa,from about 140 aa to about 150 aa, from about 150 aa to about 160 aa,from about 160 aa to about 170 aa, from about 170 aa to about 180 aa,from about 180 aa to about 190 aa, or from about 190 aa to about 200 aaof any of the following amino acid sequences:

ABI1: (SEQ ID NO: 90) MEEVSPAIAGPFRPFSETQMDFTGIRLGKGYCNNQYSNQDSENGDLMVSLPETSSCSVSGSHGSESRKVLISRINSPNLNMKESAAADIVVVDISAGDEINGSDITSEKKMISRTESRSLFEFKSVPLYGFTSICGRRPEMEDAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYCRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAVVFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLAMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKKNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLKPRRKLKSKPLN. ABI2: (SEQ ID NO: 91)MDEVSPAVAVPFRPFTDPHAGLRGYCNGESRVTLPESSCSGDGAMKDSSFEINTRQDSLTSSSSAMAGVDISAGDEINGSDEFDPRSMNQSEKKVLSRTESRSLFEFKCVPLYGVTSICGRRPEMEDSVSTIPRFLQVSSSSLLDGRVTNGFNPHLSAHFFGVYDGHGGSQVANYCRERMHLALTEEIVKEKPEFCDGDTWQEKWKKALFNSFMRVDSEIETVAHAPETVGSTSVVAVVFPTHIFVANCGDSRAVLCRGKTPLALSVDHKPDRDDEAARIEAAGGKVIRWNGARVFGVLAMSRSIGDRYLKPSVIPDPEVTSVRRVKEDDCLILASDGLWDVMTNEEVCDLARKRILLWHKKNAMAGEALLPAEKRGEGKDPAAMSAAEYLSKMALQKGSKDNISVVVVDLKGIRKFKSKSLN.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from a Cry2 protein (also known as cryptochrome 2). For examplea member of a subject dimer (e.g., a dimerizer-binding pair) can bederived from Cry2 proteins from any organism (e.g., a plant) such as,but not limited to, those of Arabidopsis thaliana. For example, asuitable dimerizer-binding pair member can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or 100%amino acid sequence identity to a contiguous stretch of from about 100amino acids to about 110 amino acids (aa), from about 110 aa to about115 aa, from about 115 aa to about 120 aa, from about 120 aa to about130 aa, from about 130 aa to about 140 aa, from about 140 aa to about150 aa, from about 150 aa to about 160 aa, from about 160 aa to about170 aa, from about 170 aa to about 180 aa, from about 180 aa to about190 aa, or from about 190 aa to about 200 aa of any of the followingamino acid sequences:

Cry2 (Arabidopsis thaliana) (SEQ ID NO: 92)MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSD QITTSLGKNGCK.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from the CIB1 Arabidopsis thaliana protein (also known astranscription factor bHLH63). For example, a suitable dimer (e.g., adimerizer-binding pair) member can comprise an amino acid sequencehaving at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, or 100% aminoacid sequence identity to a contiguous stretch of from about 100 aminoacids to about 110 amino acids (aa), from about 110 aa to about 115 aa,from about 115 aa to about 120 aa, from about 120 aa to about 130 aa,from about 130 aa to about 140 aa, from about 140 aa to about 150 aa,from about 150 aa to about 160 aa, from about 160 aa to about 170 aa,from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, orfrom about 190 aa to about 200 aa of the following amino acid sequence:

(SEQ ID NO: 93) MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIHVRARRGQATDSHSIAERVRREKISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEFLSMKLAIVNPRPDFDMDDIFAKEVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYLHVNPMQQVNTSSDPLSCFNNGEAPSMWDSHVQNLYGNLGV.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from the GAI Arabidopsis thaliana protein (also known asGibberellic Acid Insensitive, and DELLA protein GAI). For example, asuitable dimerizer-binding pair member can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or 100%amino acid sequence identity to a contiguous stretch of from about 100amino acids to about 110 amino acids (aa), from about 110 aa to about115 aa, from about 115 aa to about 120 aa, from about 120 aa to about130 aa, from about 130 aa to about 140 aa, from about 140 aa to about150 aa, from about 150 aa to about 160 aa, from about 160 aa to about170 aa, from about 170 aa to about 180 aa, from about 180 aa to about190 aa, or from about 190 aa to about 200 aa of the following amino acidsequence:

(SEQ ID NO: 94) MKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEVMMSNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLNPPSSNAEYDLKAIPGDAILNQFAIDSASSSNQGGGGDTYTTNKRLKCSNGVVETTTATAESTRHVVLVDSQENGVRLVHALLACAEAVQKENLTVAEALVKQIGFLAVSQIGAMRKVATYFAEALARRIYRLSPSQSPIDHSLSDTLQMHFYETCPYLKFAHFTANQAILEAFQGKKRVHVIDFSMSQGLQWPALMQALALRPGGPPVFRLTGIGPPAPDNFDYLHEVGCKLAHLAEAIHVEFEYRGFVANTLADLDASMLELRPSEIESVAVNSVFELHKLLGRPGAIDKVLGVVNQIKPEIFTVVEQESNHNSPIFLDRFTESLHYYSTLFDSLEGVPSGQDKVMSEVYLGKQICNVVACDGPDRVERHETLSQWRNRFGSAGFAAAHIGSNAFKQASMLLALFNGGEGYRVEESDGCLMLGWHTRPLIATSAWKLSTN.

In some cases, a member of a dimer (e.g., a dimerizer-binding pair) isderived from a GID1 Arabidopsis thaliana protein (also known asGibberellin receptor GID1). For example, a suitable dimer member cancomprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100% amino acid sequence identity to a contiguousstretch of from about 100 amino acids to about 110 amino acids (aa),from about 110 aa to about 115 aa, from about 115 aa to about 120 aa,from about 120 aa to about 130 aa, from about 130 aa to about 140 aa,from about 140 aa to about 150 aa, from about 150 aa to about 160 aa,from about 160 aa to about 170 aa, from about 170 aa to about 180 aa,from about 180 aa to about 190 aa, or from about 190 aa to about 200 aaof any of the following amino acid sequences:

GID1A: (SEQ ID NO: 95)MAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTANANPVDGVFSFDVLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFFHGGSFAHSSANSAIYDTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWVNSRSWLKSKKDSKVHIFLAGDSSGGNIAHNVALRAGESGIDVLGNILLNPMFGGNERTESEKSLDGKYFVTVRDRDWYWKAFLPEGEDREHPACNPFSPRGKSLEGVSFPKSLVVVAGLDLIRDWQLAYAEGLKKAGQEVKLMHLEKATVGFYLLPNNNHFHNVMDEISAFVNAEC. GID1B: (SEQ ID NO: 96)MAGGNEVNLNECKRIVPLNTWVLISNFKLAYKVLRRPDGSFNRDLAEFLDRKVPANSFPLDGVFSFDHVDSTTNLLTRIYQPASLLHQTRHGTLELTKPLSTTEIVPVLIFFHGGSFTHSSANSAIYDTFCRRLVTICGVVVVSVDYRRSPEHRYPCAYDDGWNALNWVKSRVWLQSGKDSNVYVYLAGDSSGGNIAHNVAVRATNEGVKVLGNILLHPMFGGQERTQSEKTLDGKYFVTIQDRDWYWRAYLPEGEDRDHPACNPFGPRGQSLKGVNFPKSLVVVAGLDLVQDWQLAYVDGLKKTGLEVNLLYLKQATIGFYFLPNNDHFHCLMEELNKFVHSIEDSQSK SSPVLLTP GID1C: (SEQID NO: 97) MAGSEEVNLIESKTVVPLNTWVLISNFKLAYNLLRRPDGTFNRHLAEFLDRKVPANANPVNGVFSFDVIIDRQTNLLSRVYRPADAGTSPSITDLQNPVDGEIVPVIVFFHGGSFAHSSANSAIYDTLCRRLVGLCGAVVVSVNYRRAPENRYPCAYDDGWAVLKWVNSSSWLRSKKDSKVRIFLAGDSSGGNIVHNVAVRAVESRIDVLGNILLNPMFGGTERTESEKRLDGKYFVTVRDRDWYWRAFLPEGEDREHPACSPFGPRSKSLEGLSFPKSLVVVAGLDLIQDWQLKYAEGLKKAGQEVKLLYLEQATIGFYLLPNNNHFHTVMDEIAAFVNAECQ.

Dimerizers

Dimerizers (“dimerizing agents) that can provide for dimerization of afirst member of a dimerizer-binding pair and a second member of adimerizer-binding pair include, e.g. (where the dimerizer is inparentheses following the dimerizer-binding pair:

-   a) FKBP and FKBP (rapamycin);-   b) FKBP and CnA (rapamycin);-   c) FKBP and cyclophilin (rapamycin);-   d) FKBP and FRG (rapamycin);-   e) GyrB and GyrB (coumermycin);-   f) DHFR and DHFR (methotrexate);-   g) DmrB and DmrB (AP20187);-   h) PYL and ABI (abscisic acid);-   i) Cry2 and CIB1 (blue light); and-   j) GAI and GID1 (gibberellin).

As noted above, rapamycin can serve as a dimerizer. Alternatively, arapamycin derivative or analog can be used. See, e.g., WO96/41865; WO99/36553; WO 01/14387; and Ye et al (1999) Science 283:88-91. Forexample, analogs, homologs, derivatives and other compounds relatedstructurally to rapamycin (“rapalogs”) include, among others, variantsof rapamycin having one or more of the following modifications relativeto rapamycin: demethylation, elimination or replacement of the methoxyat C7, C42 and/or C29; elimination, derivatization or replacement of thehydroxy at C13, C43 and/or C28; reduction, elimination or derivatizationof the ketone at C14, C24 and/or C30; replacement of the 6-memberedpipecolate ring with a 5-membered prolyl ring; and alternativesubstitution on the cyclohexyl ring or replacement of the cyclohexylring with a substituted cyclopentyl ring. Additional information ispresented in, e.g., U.S. Pat. Nos. 5,525,610; 5,310,903 5,362,718; and5,527,907. Selective epimerization of the C-28 hydroxyl group has beendescribed; see, e.g., WO 01/14387. Additional synthetic dimerizingagents suitable for use as an alternative to rapamycin include thosedescribed in U.S. Patent Publication No. 2012/0130076.

Rapamycin has the structure:

Suitable rapalogs include, e.g.,

Also suitable as a rapalog is a compound of the formula:

where n is 1 or 2; R²⁸ and R⁴³ are independently H, or a substituted orunsubstituted aliphatic or acyl moiety; one of R^(7a) and R^(7b) is Hand the other is halo, R^(A), OR^(A), SR^(A), —OC(O)R^(A),—OC(O)NR^(A)R^(B), —NR^(A)R^(B), —NR^(B)C(OR)R^(A), NR^(B)C(O)OR^(A),—NR^(B)SO₂R^(A), or NR^(B)SO₂NR^(A)R^(B′); or R^(7a) and R^(7b), takentogether, are H in the tetraene moiety:

where R^(A) is H or a substituted or unsubstituted aliphatic,heteroaliphatic, aryl, or heteroaryl moiety and where R^(B) and R^(B′)are independently H, OH, or a substituted or unsubstituted aliphatic,heteroaliphatic, aryl, or heteroaryl moiety.

As noted above, coumermycin can serve as a dimerizing agent.Alternatively, a coumermycin analog can be used. See, e.g., Farrar etal. (1996) Nature 383:178-181; and U.S. Pat. No. 6,916,846.

As noted above, in some cases, the dimerizing agent is methotrexate,e.g., a non-cytotoxic, homo-bifunctional methotrexate dimer. See, e.g.,U.S. Pat. No. 8,236,925.

Intracellular Signaling Domain

Intracellular signaling domains suitable for use in a CAR of the presentdisclosure include any desired signaling domain that provides a distinctand detectable signal (e.g., increased production of one or morecytokines by the cell; change in transcription of a target gene; changein activity of a protein; change in cell behavior, e.g., cell death;cellular proliferation; cellular differentiation; cell survival;modulation of cellular signaling responses; etc.) in response toactivation of the CAR (i.e., activated by antigen and dimerizing agent).In some embodiments, the intracellular signaling domain includes atleast one (e.g., one, two, three, four, five, six, etc.) ITAM motifs asdescribed below. In some embodiments, the intracellular signaling domainincludes DAP10/CD28 type signaling chains. In some embodiments, theintracellular signaling domain is not covalently attached to themembrane bound CAR, but is instead diffused in the cytoplasm.

ITAM

Intracellular signaling domains suitable for use in a CAR of the presentdisclosure include immunoreceptor tyrosine-based activation motif(ITAM)-containing intracellular signaling polypeptides. An ITAM motif isYX₁X₂L/I, where X₁ and X₂ are independently any amino acid (SEQ IDNO:130). In some cases, the intracellular signaling domain of a subjectCAR comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motifis repeated twice in an intracellular signaling domain, where the firstand second instances of the ITAM motif are separated from one another by6 to 8 amino acids, e.g., (YX₁X₂L/I)(X₃)_(n)(YX₁X₂L/I), where n is aninteger from 6 to 8, and each of the 6-8 X₃ can be any amino acid (SEQID NO:131). In some cases, the intracellular signaling domain of asubject CAR comprises 3 ITAM motifs.

A suitable intracellular signaling domain can be an ITAMmotif-containing portion that is derived from a polypeptide thatcontains an ITAM motif. For example, a suitable intracellular signalingdomain can be an ITAM motif-containing domain from any ITAMmotif-containing protein. Thus, a suitable intracellular signalingdomain need not contain the entire sequence of the entire protein fromwhich it is derived. Examples of suitable ITAM motif-containingpolypeptides include, but are not limited to: DAP12; FCER1G (Fc epsilonreceptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3gamma); CD3Z (CD3 zeta); and CD79A (antigen receptor complex-associatedprotein alpha chain).

In some cases, the intracellular signaling domain is derived from DAP12(also known as TYROBP; TYRO protein tyrosine kinase binding protein;KARAP; PLOSL; DNAX-activation protein 12; KAR-associated protein; TYROprotein tyrosine kinase-binding protein; killer activating receptorassociated protein; killer-activating receptor-associated protein;etc.). For example, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to any of the following amino acid sequences (4 isoforms):

(SEQ ID NO: 98) MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP YQEL QGQRSD V YSDL NTQRPYYK;(SEQ ID NO: 99) MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP YQEL QGQRSDV YSDL NTQRPYYK; (SEQID NO: 100) MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESP YQEL QGQRSDV YSDL NTQRPY YK; or (SEQ IDNO: 101) MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESP YQEL QGQRSDV YSDL NTQRPYY K,where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length DAP12 aminoacid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence:

(SEQ ID NO: 102) ESP YQEL QGQRSDV YSDL NTQ,where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from FCER1G(also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptorgamma-chain; fc-epsilon RI-gamma; fcRgamma; fceRI gamma; high affinityimmunoglobulin epsilon receptor subunit gamma; immunoglobulin Ereceptor, high affinity, gamma chain; etc.). For example, a suitableintracellular signaling domain polypeptide can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or 100%amino acid sequence identity to the following amino acid sequence:

(SEQ ID NO: 103) MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGV YTGL STRNQET YETL KHEKPPQ,where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length FCER1Gamino acid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence:

(SEQ ID NO: 104) DGV YTGL STRNQET YETL KHE,where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA;T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, deltapolypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 deltachain; T-cell surface glycoprotein CD3 delta chain; etc.). For example,a suitable intracellular signaling domain polypeptide can comprise anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, or 100%, amino acid sequence identity to a contiguous stretch offrom about 100 amino acids to about 110 amino acids (aa), from about 110aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aato about 130 aa, from about 130 aa to about 140 aa, from about 140 aa toabout 150 aa, or from about 150 aa to about 170 aa, of either of thefollowing amino acid sequences (2 isoforms):

(SEQ ID NO: 105) MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQV YQ PL RDRDDAQ YSHLGGNWARNK or (SEQ ID NO: 106)MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLR NDQV YQPL RDRDDAQYSHL GGNWARNK,where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 deltaamino acid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence:

(SEQ ID NO: 107) DQV YQPL RDRDDAQ YSHL GGN,where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 epsilon chain (also known as CD3e, T-cellsurface antigen T3/Leu-4 epsilon chain, T-cell surface glycoprotein CD3epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). For example, asuitable intracellular signaling domain polypeptide can comprise anamino acid sequence having at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, or 100%, amino acid sequence identity to a contiguous stretch offrom about 100 amino acids to about 110 amino acids (aa), from about 110aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aato about 130 aa, from about 130 aa to about 140 aa, from about 140 aa toabout 150 aa, or from about 150 aa to about 205 aa, of the followingamino acid sequence:

(SEQ ID NO: 108) MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPD YEPI RKGQRDL YS GL NQRRI,where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 epsilonamino acid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence:

(SEQ ID NO: 109) NPD YEPI RKGQRDL YSGL NQR,where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 gamma chain (also known as CD3G, T-cellreceptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3complex), etc.). For example, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to a contiguous stretch of from about 100 amino acids to about110 amino acids (aa), from about 110 aa to about 115 aa, from about 115aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aato about 140 aa, from about 140 aa to about 150 aa, or from about 150 aato about 180 aa, of the following amino acid sequence:

(SEQ ID NO: 110) MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDK QTLLPNDQL YQPLKDREDDQ YSHL QGNQLRRN,where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 gammaamino acid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence:

(SEQ ID NO: 111) DQL YQPL KDREDDQ YSHL QGN,where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptorT3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). Forexample, a suitable intracellular signaling domain polypeptide cancomprise an amino acid sequence having at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, or 100%, amino acid sequence identity to acontiguous stretch of from about 100 amino acids to about 110 aminoacids (aa), from about 110 aa to about 115 aa, from about 115 aa toabout 120 aa, from about 120 aa to about 130 aa, from about 130 aa toabout 140 aa, from about 140 aa to about 150 aa, or from about 150 aa toabout 160 aa, of either of the following amino acid sequences (2isoforms):

(SEQ ID NO: 112) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL YNEL NLGRREE YDVL DKRRGRDPEMGGKP RRKNPQEGL YNELQKDKMAEA YSEI GMKGERRRGKGHDGL YQGL STATKD T YDAL HMQALPPR or (SEQ ID NO:113) MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQL YNEL NLGRREE YDVL DKRRGRDPEMGGKP QRRKNPQEGL YNELQKDKMAEA YSEI GMKGERRRGKGHDGL YQGL STATK DT YDAL HMQALPPR,where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD3 zetaamino acid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to any of the following amino acid sequences:

(SEQ ID NO: 18) RVKFSRSADAPAYQQGQNQL YNEL NLGRREE YDVL DKRRGRDPEMGGKPRRKNPQEGL YNEL QKDKMAEA YSEI GMKGERRRGKGHDGL YQGL STATKDT YDAL HMQALPPR;(SEQ ID NO: 114) NQL YNEL NLGRREE YDVL DKR; (SEQ ID NO: 115) EGL YNELQKDKMAEA YSEI GMK; or (SEQ ID NO: 116) DGL YQGL STATKDT YDAL HMQ,where the ITAM motifs are in bold and are underlined.

In some cases, the intracellular signaling domain is derived from CD79A(also known as B-cell antigen receptor complex-associated protein alphachain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membraneglycoprotein; ig-alpha; membrane-bound immunoglobulin-associatedprotein; surface IgM-associated protein; etc.). For example, a suitableintracellular signaling domain polypeptide can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or100%, amino acid sequence identity to a contiguous stretch of from about100 amino acids to about 110 amino acids (aa), from about 110 aa toabout 115 aa, from about 115 aa to about 120 aa, from about 120 aa toabout 130 aa, from about 130 aa to about 150 aa, from about 150 aa toabout 200 aa, or from about 200 aa to about 220 aa, of either of thefollowing amino acid sequences (2 isoforms):

(SEQ ID NO: 117) MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENL YEGL NLDDCSM YE DISRGLQGTYQDVGSLNIGDVQLEKP; or (SEQ ID NO: 118)MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP,where the ITAM motifs are in bold and are underlined.

Likewise, a suitable intracellular signaling domain polypeptide cancomprise an ITAM motif-containing portion of the full length CD79A aminoacid sequence. Thus, a suitable intracellular signaling domainpolypeptide can comprise an amino acid sequence having at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or 100%, amino acid sequenceidentity to the following amino acid sequence:

(SEQ ID NO: 119) ENL YEGL NLDDCSM YEDI SRG,where the ITAM motifs are in bold and are underlined.

DAP10/CD28

Intracellular signaling domains suitable for use in a CAR of the presentdisclosure include a DAP10/CD28 type signaling chain.

An example of a DAP10 signaling chain is the amino acid sequence is:

(SEQ ID NO: 120) RPRRSPAQDGKV YINM PGRG.In some embodiments, a suitable intracellular signaling domain comprisesan amino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, amino acidsequence identity to the entire length of the amino acid sequence

(SEQ ID NO: 120) RPRRSPAQDGKV YINM PGRG.

An example of a CD28 signaling chain is the amino acid sequence is

(SEQ ID NO: 121) FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD YMNM TPRRPGPTRKHYQPYAPPRDFAAYRS.In some embodiments, a suitable intracellular signaling domain comprisesan amino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98%, or at least about 99%, amino acidsequence identity to the entire length of the amino acid sequence

(SEQ ID NO: 121) FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD YMNM TPRRPGPTRKHYQPYAPPRDFAAYRS.

Intracellular signaling domains suitable for use in a CAR of the presentdisclosure include a ZAP70 polypeptide, e.g., a polypeptide comprisingan amino acid sequence having at least about 85%, at least about 90%, atleast about 95%, at least about 98%, at least about 99%, or 100%, aminoacid sequence identity to a contiguous stretch of from about 300 aminoacids to about 400 amino acids, from about 400 amino acids to about 500amino acids, or from about 500 amino acids to 619 amino acids, of thefollowing amino acid sequence:

(SEQ ID NO: 36) MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSL ASKVEGPPGSTQKAEAACA.

Additional Sequences

The first and/or the second polypeptide of a subject CAR can furtherinclude one or more additional polypeptide domains, where such domainsinclude, but are not limited to, a signal sequence; an epitope tag; anaffinity domain; and a polypeptide that produces a detectable signal.

Signal Sequences

Signal sequences that are suitable for use in a subject CAR, e.g., inthe first polypeptide of a subject CAR, include any eukaryotic signalsequence, including a naturally-occurring signal sequence, a synthetic(e.g., man-made) signal sequence, etc.

Epitope Tag

Suitable epitope tags include, but are not limited to, hemagglutinin(HA; e.g., YPYDVPDYA (SEQ ID NO:122); FLAG (e.g., DYKDDDDK (SEQ IDNO:123); c-myc (e.g., EQKLISEEDL; SEQ ID NO:4), and the like.

Affinity Domain

Affinity domains include peptide sequences that can interact with abinding partner, e.g., such as one immobilized on a solid support,useful for identification or purification. DNA sequences encodingmultiple consecutive single amino acids, such as histidine, when fusedto the expressed protein, may be used for one-step purification of therecombinant protein by high affinity binding to a resin column, such asnickel sepharose. Exemplary affinity domains include His5 (HHHHH) (SEQID NO:124), HisX6 (HHHHHH) (SEQ ID NO:125), C-myc (EQKLISEEDL) (SEQ IDNO:4), Flag (DYKDDDDK) (SEQ ID NO:123), StrepTag (WSHPQFEK) (SEQ IDNO:126), hemagluttinin, e.g., HA Tag (YPYDVPDYA) (SEQ ID NO:122), GST,thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:127),Phe-His-His-Thr (SEQ ID NO:128), chitin binding domain, S-peptide, T7peptide, SH2 domain, C-end RNA tag, WEAAAREACCRECCARA (SEQ ID NO:129),metal binding domains, e.g., zinc binding domains or calcium bindingdomains such as those from calcium-binding proteins, e.g., calmodulin,troponin C, calcineurin B, myosin light chain, recoverin, S-modulin,visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpainlarge-subunit, S100 proteins, parvalbumin, calbindin D9K, calbindinD28K, and calretinin, inteins, biotin, streptavidin, MyoD, Id, leucinezipper sequences, and maltose binding protein.

Detectable Signal-Producing Polypeptides

Suitable detectable signal-producing proteins include, e.g., fluorescentproteins; enzymes that catalyze a reaction that generates a detectablesignal as a product; and the like.

Suitable fluorescent proteins include, but are not limited to, greenfluorescent protein (GFP) or variants thereof, blue fluorescent variantof GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescentvariant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhancedYFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine,GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP),destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet,mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2,t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP,Kaede protein and kindling protein, Phycobiliproteins andPhycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrinand Allophycocyanin. Other examples of fluorescent proteins includemHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry,mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat.Methods 2:905-909), and the like. Any of a variety of fluorescent andcolored proteins from Anthozoan species, as described in, e.g., Matz etal. (1999) Nature Biotechnol. 17:969-973, is suitable for use.

Suitable enzymes include, but are not limited to, horse radishperoxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL),glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase,β-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase,glucose oxidase (GO), and the like.

Recombination of Sequences

In certain instances, sequences of the polypeptides of a CAR, e.g., CARdomains, may be rearranged or deleted in a cell through the use ofsite-specific recombination technology. In certain embodiments, thecellular activation-related response to a particular CAR can be changedby site-specific recombination, e.g., a first intracellular signalingdomain of a CAR eliciting a first activation-related response may beexchanged for a second intracellular signaling domain eliciting a secondactivation-related response. In certain instances, the response to aparticular dimerizer of a CAR can be changed by site-specificrecombination, e.g., a first dimerizer-binding pair causing thedimerization of a CAR in the presence of a first dimerizer may beexchanged for a second dimerizer-binding pair causing the dimerizationof the CAR in the presence of a second dimerizer. As will be clear toone skilled in the art, site-specific recombination can be used in acell to exchange any domain or sequence of a CAR with any other domainor sequence as disclosed herein. As will also be clear to one skilled inthe art, site-specific recombination can be used in a cell to delete anydomain or sequence of a CAR. Such exchange and excision of sequences anddomains is known in the art, see, e.g., domain switching insignalobodies as described in Tone et al. (2013) Biotechnology andBioengineering, 3219-3226, the disclosure of which is disclosed hereinby reference. Mechanisms and requirements for performing site-specificrecombination in vivo are also well known in the art, see, e.g.,Grindley et al. (2006) Annual Review of Biochemistry, 567-605 and Tropp(2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, Mass.),the disclosures of which are incorporated herein by reference.

Nucleic Acids

The present disclosure provides a nucleic acid that comprises anucleotide sequence encoding the first and/or the second polypeptide ofa heterodimeric, conditionally active CAR of the present disclosure. Anucleic acid comprising a nucleotide sequence encoding the first and/orthe second polypeptide of a heterodimeric, conditionally active CAR ofthe present disclosure will in some embodiments be DNA, including, e.g.,a recombinant expression vector. A nucleic acid comprising a nucleotidesequence encoding the first and/or the second polypeptide of aheterodimeric, conditionally active CAR of the present disclosure willin some embodiments be RNA, e.g., in vitro synthesized RNA.

In some cases, a nucleic acid of the present disclosure comprises anucleotide sequence encoding only the first polypeptide (and not thesecond polypeptide) of a heterodimeric, conditionally active CAR of thepresent disclosure. In some cases, a nucleic acid of the presentdisclosure comprises a nucleotide sequence encoding only the secondpolypeptide (and not the first polypeptide) of a heterodimeric,conditionally active CAR of the present disclosure. In some cases, anucleic acid of the present disclosure comprises a nucleotide sequenceencoding both the first polypeptide and the second polypeptide of aheterodimeric, conditionally active CAR of the present disclosure.

In some cases, a subject nucleic acid provides for production of a CARof the present disclosure, e.g., in a mammalian cell. In other cases, asubject nucleic acid provides for amplification of the CAR-encodingnucleic acid.

A nucleotide sequence encoding the first and/or the second polypeptideof a CAR of the present disclosure can be operably linked to atranscriptional control element, e.g., a promoter, and enhancer, etc.

Suitable promoter and enhancer elements are known in the art. Forexpression in a bacterial cell, suitable promoters include, but are notlimited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression ina eukaryotic cell, suitable promoters include, but are not limited to,light and/or heavy chain immunoglobulin gene promoter and enhancerelements; cytomegalovirus immediate early promoter; herpes simplex virusthymidine kinase promoter; early and late SV40 promoters; promoterpresent in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters.

Suitable reversible promoters, including reversible inducible promotersare known in the art. Such reversible promoters may be isolated andderived from many organisms, e.g., eukaryotes and prokaryotes.Modification of reversible promoters derived from a first organism foruse in a second organism, e.g., a first prokaryote and a second aeukaryote, a first eukaryote and a second a prokaryote, etc., is wellknown in the art. Such reversible promoters, and systems based on suchreversible promoters but also comprising additional control proteins,include, but are not limited to, alcohol regulated promoters (e.g.,alcohol dehydrogenase I (alcA) gene promoter, promoters responsive toalcohol transactivator proteins (AlcR), etc.), tetracycline regulatedpromoters, (e.g., promoter systems including TetActivators, TetON,TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoidreceptor promoter systems, human estrogen receptor promoter systems,retinoid promoter systems, thyroid promoter systems, ecdysone promotersystems, mifepristone promoter systems, etc.), metal regulated promoters(e.g., metallothionein promoter systems, etc.), pathogenesis-relatedregulated promoters (e.g., salicylic acid regulated promoters, ethyleneregulated promoters, benzothiadiazole regulated promoters, etc.),temperature regulated promoters (e.g., heat shock inducible promoters(e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), lightregulated promoters, synthetic inducible promoters, and the like.

In some instances, the locus or construct or transgene containing thesuitable promoter is irreversibly switched through the induction of aninducible system. Suitable systems for induction of an irreversibleswitch are well known in the art, e.g., induction of an irreversibleswitch may make use of a Cre-lox-mediated recombination (see, e.g.,Fuhrmann-Benzakein, et al., PNAS (2000) 28:e99, the disclosure of whichis incorporated herein by reference). Any suitable combination ofrecombinase, endonuclease, ligase, recombination sites, etc. known tothe art may be used in generating an irreversibly switchable promoter.Methods, mechanisms, and requirements for performing site-specificrecombination, described elsewhere herein, find use in generatingirreversibly switched promoters and are well known in the art, see,e.g., Grindley et al. (2006) Annual Review of Biochemistry, 567-605 andTropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury,Mass.), the disclosures of which are incorporated herein by reference.

In some cases, the promoter is a CD8 cell-specific promoter, a CD4cell-specific promoter, a neutrophil-specific promoter, or anNK-specific promoter. For example, a CD4 gene promoter can be used; see,e.g., Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739; andMarodon et al. (2003) Blood 101:3416. As another example, a CD8 genepromoter can be used. NK cell-specific expression can be achieved by useof an Ncrl (p46) promoter; see, e.g., Eckelhart et al. (2011) Blood117:1565.

In some embodiments, e.g., for expression in a yeast cell, a suitablepromoter is a constitutive promoter such as an ADH1 promoter, a PGK1promoter, an ENO promoter, a PYK1 promoter and the like; or aregulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2promoter, a PHO5 promoter, a CUP1 promoter, a GALT promoter, a MET25promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1promoter, and AOX1 (e.g., for use in Pichia). Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Suitable promoters for use in prokaryotic host cells include, but arenot limited to, a bacteriophage T7 RNA polymerase promoter; a trppromoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tachybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lacpromoter; a trc promoter; a tac promoter, and the like; an araBADpromoter; in vivo regulated promoters, such as an ssaG promoter or arelated promoter (see, e.g., U.S. Patent Publication No. 20040131637), apagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991: 173(1): 86-93;Alpuche-Aranda et al., PNAS, 1992; 89(21): 10079-83), a nirB promoter(Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see,e.g., Dunstan et al. (1999) Infect. Immun. 67:5133-5141; McKelvie et al.(2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol.10:888-892); a sigma70 promoter, e.g., a consensus sigma70 promoter(see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); astationary phase promoter, e.g., a dps promoter, an spy promoter, andthe like; a promoter derived from the pathogenicity island SPI-2 (see,e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al.(2002) Infect. Immun. 70:1087-1096); an rpsM promoter (see, e.g.,Valdivia and Falkow (1996). Mol. Microbiol. 22:367); a tet promoter(see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. andHeinemann, U. (eds), Topics in Molecular and Structural Biology,Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp.143-162); an SP6 promoter (see, e.g., Melton et al. (1984) Nucl. AcidsRes. 12:7035); and the like. Suitable strong promoters for use inprokaryotes such as Escherichia coli include, but are not limited toTrc, Tac, T5, T7, and P_(Lambda). Non-limiting examples of operators foruse in bacterial host cells include a lactose promoter operator (LacIrepressor protein changes conformation when contacted with lactose,thereby preventing the Lad repressor protein from binding to theoperator), a tryptophan promoter operator (when complexed withtryptophan, TrpR repressor protein has a conformation that binds theoperator; in the absence of tryptophan, the TrpR repressor protein has aconformation that does not bind to the operator), and a tac promoteroperator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci.U.S.A. 80:21-25).

A nucleotide sequence encoding a subject CAR can be present in anexpression vector and/or a cloning vector. Where a subject CAR comprisestwo separate polypeptides, nucleotide sequences encoding the twopolypeptides can be cloned in the same or separate vectors. Anexpression vector can include a selectable marker, an origin ofreplication, and other features that provide for replication and/ormaintenance of the vector. Suitable expression vectors include, e.g.,plasmids, viral vectors, and the like.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant constructs. The following vectors are provided byway of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK,pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif.,USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia,Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG(Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Suitable expression vectorsinclude, but are not limited to, viral vectors (e.g. viral vectors basedon vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., InvestOpthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., HGene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see,e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al.,Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594,1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989)63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte etal., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; humanimmunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23,1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector(e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derivedfrom retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,avian leukosis virus, human immunodeficiency virus, myeloproliferativesarcoma virus, and mammary tumor virus); and the like.

As noted above, in some embodiments, a nucleic acid comprising anucleotide sequence encoding the first and/or the second polypeptide ofa heterodimeric, conditionally active CAR of the present disclosure willin some embodiments be RNA, e.g., in vitro synthesized RNA. Methods forin vitro synthesis of RNA are known in the art; any known method can beused to synthesize RNA comprising a nucleotide sequence encoding thefirst and/or the second polypeptide of a heterodimeric, conditionallyactive CAR of the present disclosure. Methods for introducing RNA into ahost cell are known in the art. See, e.g., Zhao et al. (2010) CancerRes. 15:9053. Introducing RNA comprising a nucleotide sequence encodingthe first and/or the second polypeptide of a heterodimeric,conditionally active CAR of the present disclosure into a host cell canbe carried out in vitro or ex vivo or in vivo. For example, a host cell(e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporatedin vitro or ex vivo with RNA comprising a nucleotide sequence encodingthe first and/or the second polypeptide of a heterodimeric,conditionally active CAR of the present disclosure.

Cells

The present disclosure provides a mammalian cell that is geneticallymodified to produce a heterodimeric, conditionally active CAR of thepresent disclosure.

Suitable mammalian cells include primary cells and immortalized celllines. Suitable mammalian cell lines include human cell lines, non-humanprimate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.Suitable mammalian cell lines include, but are not limited to, HeLacells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHOcells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCCNo. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658),Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse Lcells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No.CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g.,NKL, NK92, and YTS), and the like.

In some instances, the cell is not an immortalized cell line, but isinstead a cell (e.g., a primary cell) obtained from an individual. Forexample, in some cases, the cell is an immune cell obtained from anindividual. As an example, the cell is a T lymphocyte obtained from anindividual. As another example, the cell is a cytotoxic cell obtainedfrom an individual. As another example, the cell is a stem cell orprogenitor cell obtained from an individual.

Methods of Activating an Immune Cell

The present disclosure provides methods of activating an immune cell invitro, in vivo, or ex vivo. The methods generally involve contacting animmune cell (in vitro, in vivo, or ex vivo) with a dimerizing agent andan antigen, where the immune cell is genetically modified to produce aheterodimeric, conditionally active CAR of the present disclosure. Inthe presence of the dimerizing agent and the antigen, the heterodimeric,conditionally active CAR dimerizes and activates the immune cell,thereby producing an activated immune cell. Immune cells include, e.g.,a cytotoxic T lymphocyte, an NK cell, a CD4⁺ T cell, a T regulatory(Treg) cell, etc.

Contacting the genetically modified immune cell (e.g., a T lymphocyte,an NK cell) with a dimerizing agent and a second member of a specificbinding pair (e.g., an antigen, a ligand, a receptor) can increaseproduction of a cytokine by the immune cell by at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 40%, at least about 50%, at least about 75%, atleast about 2-fold, at least about 2.5-fold, at least about 5-fold, atleast about 10-fold, or more than 10-fold, compared with the amount ofcytokine produced by the immune cell in the absence of the second memberof a specific binding pair and/or the dimerizing agent. Cytokines whoseproduction can be increased include, but are not limited to, IL-2 andIFN-γ.

Contacting the genetically modified immune cell (e.g., a T lymphocyte,an NK cell) with a dimerizing agent and an antigen can increaseproduction of a cytokine by the immune cell by at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 40%, at least about 50%, at least about 75%, atleast about 2-fold, at least about 2.5-fold, at least about 5-fold, atleast about 10-fold, or more than 10-fold, compared with the amount ofcytokine produced by the immune cell in the absence of the antigenand/or the dimerizing agent. Cytokines whose production can be increasedinclude, but are not limited to, IL-2 and IFN-γ.

Contacting a genetically modified cytotoxic cell (e.g., cytotoxic Tlymphocyte) with a dimerizing agent and a second member of a specificbinding pair (e.g., an antigen, a ligand, a receptor) can increasecytotoxic activity of the cytotoxic cell by at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 75%, at leastabout 2-fold, at least about 2.5-fold, at least about 5-fold, at leastabout 10-fold, or more than 10-fold, compared to the cytotoxic activityof the cytotoxic cell in the absence of the dimerizing agent.

Contacting a genetically modified cytotoxic cell (e.g., cytotoxic Tlymphocyte) with a dimerizing agent and an antigen can increasecytotoxic activity of the cytotoxic cell by at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 75%, at leastabout 2-fold, at least about 2.5-fold, at least about 5-fold, at leastabout 10-fold, or more than 10-fold, compared to the cytotoxic activityof the cytotoxic cell in the absence of the dimerizing agent.

In other embodiments, e.g., depending on the host immune cell,contacting a genetically modified host cell with a dimerizing agent andan antigen can increase or decrease cell proliferation, cell survival,cell death, and the like.

Methods of Generating A Conditionally Activatable Cell

The present disclosure provides a method of generating a conditionallyactivatable cell. The method generally involves genetically modifying amammalian cell with an expression vector, or an RNA (e.g., in vitrotranscribed RNA), comprising nucleotide sequences encoding aheterodimeric, conditionally active CAR of the present disclosure. Thegenetically modified cell is conditionally activatable in the presenceof: a) an antigen to which the first polypeptide of the CAR binds; andb) a dimerizer (a dimerizing agent). The genetic modification can becarried out in vivo, in vitro, or ex vivo. The cell can be an immunecell (e.g., a T lymphocyte or NK cell), a stem cell, a progenitor cell,etc.

In some cases, the genetic modification is carried out ex vivo. Forexample, a T lymphocyte, a stem cell, or an NK cell is obtained from anindividual; and the cell obtained from the individual is geneticallymodified to express a CAR of the present disclosure. The geneticallymodified cell is conditionally activatable in the presence of: a) anantigen to which the first polypeptide of the CAR binds; and b) adimerizer. In some cases, the genetically modified cell is activated exvivo. In other cases, the genetically modified cell is introduced intoan individual (e.g., the individual from whom the cell was obtained);and the genetically modified cell is activated in vivo, e.g., byadministering to the individual a dimerizer. For example, where theantigen is present on the surface of a cell in the individual, there isno need to administer the antigen. The genetically modified cell comesinto contact with the antigen present on the surface of a cell in theindividual; and, upon administration to the individual of a dimerizer,the genetically modified cell is activated. For example, where thegenetically modified cell is a T lymphocyte, the genetically modifiedcell can exhibit cytotoxicity toward a cell that presents an antigen onits surface to which the CAR binds.

Treatment Methods

The present disclosure provides various treatment methods using asubject CAR.

Cytotoxicity Methods

A CAR of the present disclosure, when present in a T lymphocyte or an NKcell, can mediate cytotoxicity toward a target cell. A CAR of thepresent disclosure binds to an antigen present on a target cell, therebymediating killing of a target cell by a T lymphocyte or an NK cellgenetically modified to produce the CAR. The antigen-binding domain ofthe CAR binds to an antigen present on the surface of a target cell.

Target cells include, but are not limited to, cancer cells. Thus, thepresent disclosure provides methods of killing, or inhibiting the growthof, a target cancer cell, the method involving contacting a cytotoxicimmune effector cell (e.g., a cytotoxic T cell, or an NK cell) that isgenetically modified to produce a subject CAR, such that the Tlymphocyte or NK cell recognizes an antigen present on the surface of atarget cancer cell, and mediates killing of the target cell.

The present disclosure provides a method of treating cancer in anindividual having a cancer, the method comprising: i) geneticallymodifying T lymphocytes obtained from the individual with an expressionvector comprising nucleotide sequences encoding the heterodimeric,conditionally active CAR of the present disclosure, where theantigen-binding domain of the heterodimeric, conditionally active CAR isspecific for an epitope on a cancer cell in the individual, and wherethe genetic modification is carried out ex vivo; ii) introducing thegenetically modified T lymphocytes into the individual; and iii)administering to the individual an effective amount of a dimerizingagent, wherein the dimerizing agent induces dimerization of theheterodimeric, conditionally active CAR, wherein said dimerizationprovides for activation of the genetically modified T lymphocytes andkilling of the cancer cell, thereby treating the cancer.

Carcinomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, esophageal carcinoma, hepatocellularcarcinoma, basal cell carcinoma (a form of skin cancer), squamous cellcarcinoma (various tissues), bladder carcinoma, including transitionalcell carcinoma (a malignant neoplasm of the bladder), bronchogeniccarcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma,lung carcinoma, including small cell carcinoma and non-small cellcarcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma,pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostatecarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductalcarcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterinecarcinoma, testicular carcinoma, osteogenic carcinoma, epithelialcarcinoma, and nasopharyngeal carcinoma.

Sarcomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma,leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be amenable to therapy by a method disclosedherein include, but are not limited to, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Leukemias that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, a) chronic myeloproliferative syndromes(neoplastic disorders of multipotential hematopoietic stem cells); b)acute myelogenous leukemias (neoplastic transformation of amultipotential hematopoietic stem cell or a hematopoietic cell ofrestricted lineage potential; c) chronic lymphocytic leukemias (CLL;clonal proliferation of immunologically immature and functionallyincompetent small lymphocytes), including B-cell CLL, T-cell CLLprolymphocytic leukemia, and hairy cell leukemia; and d) acutelymphoblastic leukemias (characterized by accumulation of lymphoblasts).Lymphomas that can be treated using a subject method include, but arenot limited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin'slymphoma; non-Hodgkin's lymphoma, and the like.

Other cancers that can be amenable to treatment according to the methodsdisclosed herein include atypical meningioma (brain), islet cellcarcinoma (pancreas), medullary carcinoma (thyroid), mesenchymoma(intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver),clear cell carcinoma (kidney), and neurofibroma mediastinum.

Immunomodulatory Methods

A subject method can also be used to treat inflammatory conditions andautoimmune disease. A subject CAR is expressed in a T-helper cell or aTregs for use in an immunomodulatory method. Immunomodulatory methodsinclude, e.g., enhancing an immune response in a mammalian subjecttoward a pathogen; enhancing an immune response in a subject who isimmunocompromised; reducing an inflammatory response; reducing an immuneresponse in a mammalian subject to an autoantigen, e.g., to treat anautoimmune disease; and reducing an immune response in a mammaliansubject to a transplanted organ or tissue, to reduce organ or tissuerejection.

Where the method involves reducing an immune response to an autoantigen,the antigen used to activate the CAR is an autoantigen. Where the methodinvolves reducing an immune response to a transplanted organ or tissue,the antigen used to activate the CAR is an antigen specific to thetransplanted organ.

Formulations, Dosages, and Routes of Administration

As discussed above, a treatment method of the present disclosureinvolves administration to an individual in need thereof of an effectiveamount of a dimerizer agent, and may also involve administration of anantigen.

An “effective amount” of a dimerizer agent is in some cases an amountthat, when administered in one or more doses to an individual in needthereof, increases the level of cytotoxic activity of a T lymphocyteexpressing a subject CAR by at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about40%, at least about 50%, at least about 75%, at least about 2-fold, atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, ormore than 10-fold, compared to the cytotoxic activity of the Tlymphocyte in the absence of the dimerizing agent.

An “effective amount” of a dimerizer agent is in some cases an amountthat, when administered in one or more doses to an individual in needthereof, increases the level of cytotoxic activity of an NK cellexpressing a subject CAR by at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about40%, at least about 50%, at least about 75%, at least about 2-fold, atleast about 2.5-fold, at least about 5-fold, at least about 10-fold, ormore than 10-fold, compared to the cytotoxic activity of the NK cell inthe absence of the dimerizing agent.

An “effective amount” of a dimerizer agent is in some cases an amountthat, when administered in one or more doses to an individual in needthereof, reduces the number of cancer cells in the individual and/orreduces tumor mass in the individual, by at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 75%, or more than75%, compared to the number of cancer cells and/or tumor mass in theabsence of the dimerizing agent.

In some embodiments, an effective amount of a dimerizer is an amountthat, when administered alone (e.g., in monotherapy) or in combination(e.g., in combination therapy) with one or more additional therapeuticagents, in one or more doses, is effective to reduce one or more oftumor growth rate, cancer cell number, and tumor mass, by at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, or more, compared to the tumor growth rate, cancer cellnumber, or tumor mass in the absence of treatment with the dimerizer.

Formulations

In the subject methods, a dimerizer can be administered to the hostusing any convenient means capable of resulting in the desiredtherapeutic effect or diagnostic effect. Thus, the dimerizer can beincorporated into a variety of formulations for therapeuticadministration. More particularly, a dimerizer can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, a dimerizer can be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of a dimerizer adequate to achieve thedesired state in the subject being treated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

For oral preparations, a dimerizer can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

A dimerizer can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

Pharmaceutical compositions comprising a dimerizer are prepared bymixing the dimerizer having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, stabilizers,surfactants, buffers and/or tonicity agents. Acceptable carriers,excipients and/or stabilizers are nontoxic to recipients at the dosagesand concentrations employed, and include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid,glutathione, cysteine, methionine and citric acid; preservatives (suchas ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methylor propyl parabens, benzalkonium chloride, or combinations thereof);amino acids such as arginine, glycine, ornithine, lysine, histidine,glutamic acid, aspartic acid, isoleucine, leucine, alanine,phenylalanine, tyrosine, tryptophan, methionine, serine, proline andcombinations thereof; monosaccharides, disaccharides and othercarbohydrates; low molecular weight (less than about 10 residues)polypeptides; proteins, such as gelatin or serum albumin; chelatingagents such as EDTA; sugars such as trehalose, sucrose, lactose,glucose, mannose, maltose, galactose, fructose, sorbose, raffinose,glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid;and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, orpolyethylene glycol (PEG).

The pharmaceutical composition may be in a liquid form, a lyophilizedform or a liquid form reconstituted from a lyophilized form, wherein thelyophilized preparation is to be reconstituted with a sterile solutionprior to administration. The standard procedure for reconstituting alyophilized composition is to add back a volume of pure water (typicallyequivalent to the volume removed during lyophilization); howeversolutions comprising antibacterial agents may be used for the productionof pharmaceutical compositions for parenteral administration; see alsoChen (1992) Drug Dev Ind Pharm 18, 1311-54.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity a dimerizercalculated in an amount sufficient to produce the desired effect inassociation with a pharmaceutically acceptable diluent, carrier orvehicle. The specifications for a given dimerizer may depend on theparticular dimerizer employed and the effect to be achieved, and thepharmacodynamics associated with each dimerizer in the host.

In some embodiments, a dimerizer is formulated in a controlled releaseformulation. Sustained-release preparations may be prepared usingmethods well known in the art. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the dimerizer in which the matrices are in the formof shaped articles, e.g. films or microcapsules. Examples ofsustained-release matrices include polyesters, copolymers of L-glutamicacid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,hydrogels, polylactides, degradable lactic acid-glycolic acid copolymersand poly-D-(-)-3-hydroxybutyric acid. Possible loss of biologicalactivity may be prevented by using appropriate additives, by controllingmoisture content and by developing specific polymer matrix compositions.

Dosages

A suitable dosage can be determined by an attending physician or otherqualified medical personnel, based on various clinical factors. As iswell known in the medical arts, dosages for any one patient depend uponmany factors, including the patient's size, body surface area, age, theparticular dimerizer to be administered, sex of the patient, time, androute of administration, general health, and other drugs beingadministered concurrently. A dimerizer may be administered in amountsbetween 1 ng/kg body weight and 20 mg/kg body weight per dose, e.g.between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g. between 0.5mg/kg body weight to 5 mg/kg body weight; however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. If the regimen is a continuous infusion, it canalso be in the range of 1 μg to 10 mg per kilogram of body weight perminute.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific dimerizer, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means.

Routes of Administration

A dimerizer is administered to an individual using any available methodand route suitable for drug delivery, including in vivo and ex vivomethods, as well as systemic and localized routes of administration.

Conventional and pharmaceutically acceptable routes of administrationinclude intratumoral, peritumoral, intramuscular, intratracheal,intracranial, subcutaneous, intradermal, topical application,intravenous, intraarterial, rectal, nasal, oral, and other enteral andparenteral routes of administration. Routes of administration may becombined, if desired, or adjusted depending upon the dimerizer and/orthe desired effect. A dimerizer can be administered in a single dose orin multiple doses. In some embodiments, a dimerizer is administeredorally. In some embodiments, a dimerizer is administered via aninhalational route. In some embodiments, a dimerizer is administeredintranasally. In some embodiments, a dimerizer is administered locally.In some embodiments, a dimerizer is administered intratumorally. In someembodiments, a dimerizer is administered peritumorally. In someembodiments, a dimerizer is administered intracranially. In someembodiments, a dimerizer is administered intravenously.

The agent can be administered to a host using any available conventionalmethods and routes suitable for delivery of conventional drugs,including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, intratumoral, peritumoral, and intravenous routes, i.e.,any route of administration other than through the alimentary canal.Parenteral administration can be carried to effect systemic or localdelivery of a dimerizer. Where systemic delivery is desired,administration typically involves invasive or systemically absorbedtopical or mucosal administration of pharmaceutical preparations.

A dimerizer can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as cancer. As such, treatmentalso includes situations where the pathological condition, or at leastsymptoms associated therewith, are completely inhibited, e.g. preventedfrom happening, or stopped, e.g. terminated, such that the host nolonger suffers from the pathological condition, or at least the symptomsthat characterize the pathological condition.

In some embodiments, a dimerizer is administered by injection and/ordelivery, e.g., to a site in a brain artery or directly into braintissue. A dimerizer can also be administered directly to a target sitee.g., by direct injection, by implantation of a drug delivery devicesuch as an osmotic pump or slow release particle, by biolistic deliveryto the target site, etc.

Combination Therapy

In some embodiments, a dimerizer is administered as an adjuvant therapyto a standard cancer therapy. Standard cancer therapies include surgery(e.g., surgical removal of cancerous tissue), radiation therapy, bonemarrow transplantation, chemotherapeutic treatment, antibody treatment,biological response modifier treatment, and certain combinations of theforegoing.

Radiation therapy includes, but is not limited to, x-rays or gamma raysthat are delivered from either an externally applied source such as abeam, or by implantation of small radioactive sources.

Suitable antibodies for use in cancer treatment include, but are notlimited to, naked antibodies, e.g., trastuzumab (Herceptin), bevacizumab(Avastin™), cetuximab (Erbitux™), panitumumab (Vectibix™), Ipilimumab(Yervoy™), rituximab (Rituxan), alemtuzumab (Lemtrada™), Ofatumumab(Arzerra™), Oregovomab (OvaRex™), Lambrolizumab (MK-3475), pertuzumab(Perjeta™), ranibizumab (Lucentis™) etc., and conjugated antibodies,e.g., gemtuzumab ozogamicin (Mylortarg™), Brentuximab vedotin(Adcetris™), ⁹⁰Y-labelled ibritumomab tiuxetan (Zevalin™), ¹³¹I-labelledtositumoma (Bexxar™), etc. Suitable antibodies for use in cancertreatment include, but are not limited to, antibodies raised againsttumor-associated antigens. Such antigens include, but are not limitedto, CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, Mucins, TAG-72, CAIX,PSMA, Folate-binding protein, Gangliosides (e.g., GD2, GD3, GM2, etc.),Le^(y), VEGF, VEGFR, Integrin alpha-V-beta-3, Integrin alpha-5-beta-1,EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP,Tenascin, etc.

Biological response modifiers suitable for use in connection with themethods of the present disclosure include, but are not limited to, (1)inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors ofserine/threonine kinase activity; (3) tumor-associated antigenantagonists, such as antibodies that bind specifically to a tumorantigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6)interferon-α; (7) interferon-γ; (8) colony-stimulating factors; (9)inhibitors of angiogenesis; and (10) antagonists of tumor necrosisfactor.

Chemotherapeutic agents are non-peptidic (i.e., non-proteinaceous)compounds that reduce proliferation of cancer cells, and encompasscytotoxic agents and cytostatic agents. Non-limiting examples ofchemotherapeutic agents include alkylating agents, nitrosoureas,antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, andsteroid hormones.

Agents that act to reduce cellular proliferation are known in the artand widely used. Such agents include alkylating agents, such as nitrogenmustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, andtriazenes, including, but not limited to, mechlorethamine,cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin,chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil,pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, including, but notlimited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil(5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP),pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins),include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel(Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine;brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine,vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.;antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin,rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin andmorpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g.dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinoneglycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g.mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclicimmunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf),rapamycin, etc.; and the like.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Microtubule affecting agents that have antiproliferative activity arealso suitable for use and include, but are not limited to,allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate,natural and synthetic epothilones including but not limited to,eopthilone A, epothilone B, discodermolide; estramustine, nocodazole,and the like.

Hormone modulators and steroids (including synthetic analogs) that aresuitable for use include, but are not limited to, adrenocorticosteroids,e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide; 17α-ethinylestradiol;diethylstilbestrol, testosterone, fluoxymesterone, dromostanolonepropionate, testolactone, methylprednisolone, methyl-testosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesterone acetate,leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®.Estrogens stimulate proliferation and differentiation, thereforecompounds that bind to the estrogen receptor are used to block thisactivity. Corticosteroids may inhibit T cell proliferation.

Other chemotherapeutic agents include metal complexes, e.g. cisplatin(cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines,e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor;procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

“Taxanes” include paclitaxel, as well as any active taxane derivative orpro-drug. “Paclitaxel” (which should be understood herein to includeanalogues, formulations, and derivatives such as, for example,docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), 10-desacetylanalogs of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs ofpaclitaxel) may be readily prepared utilizing techniques known to thoseskilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253;5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267),or obtained from a variety of commercial sources, including for example,Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; orT-1912 from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term “taxane” are a variety of knownderivatives, including both hydrophilic derivatives, and hydrophobicderivatives. Taxane derivatives include, but not limited to, galactoseand mannose derivatives described in International Patent ApplicationNo. WO 99/18113; piperazino and other derivatives described in WO99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, andU.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288;sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxolderivative described in U.S. Pat. No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described inWO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Subjects Suitable for Treatment

A variety of subjects are suitable for treatment with a subject methodof treating cancer. Suitable subjects include any individual, e.g., ahuman or non-human animal who has cancer, who has been diagnosed withcancer, who is at risk for developing cancer, who has had cancer and isat risk for recurrence of the cancer, who has been treated with an agentother than a dimerizer for the cancer and failed to respond to suchtreatment, or who has been treated with an agent other than a dimerizerfor the cancer but relapsed after initial response to such treatment.

Subjects suitable for treatment with a subject immunomodulatory methodinclude individuals who have an autoimmune disorder; individuals who areorgan or tissue transplant recipients; and the like; individuals who areimmunocompromised; and individuals who are infected with a pathogen.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);i.v., intravenous(ly); and the like.

Example 1: Generation of CAR Materials and Methods

The anti-human CD19 scFv was selected as the antigen recognition domainin CARs throughout the design optimization process. FIGS. 18A and 18Bsummarize the molecular structure of each CAR consisting of twonumerically identified polypeptides. All membrane-anchored polypeptidesare di-sulfide bonded homo-dimers. The membrane-anchored polypeptidesare depicted as monomers for graphical simplicity.

Generation of CAR Constructs

Sequence encoding the anti-human CD19 scFv was cloned from a construct.The human 4-1BB co-stimulation and CD3 zeta ITAM signaling chains werecloned from cDNAs supplied by Open Biosystems. FKBP- and FRB-encodingsequences were cloned from plasmids supplied by Addgene.

Standard molecular cloning techniques (polymerase chain reaction (PCR),restriction digestion, ligation, etc.) were applied to generatelentiviral expression plasmids.

Effector and Target Cell Culturing Conditions

Human primary CD8+ T cells were isolated from anonymous donor's bloodafter apheresis (Trima residuals from Blood Centers of the Pacific, SanFrancisco, Calif.) by negative selection using RosetteSep Human CD8+ TCell Enrichment Cocktail (STEMCELL Technologies #15063) as approved byUniversity Institutional Review Board. Cells were cultured in human Tcell medium, consisting of X-VIVO15 (Lonza #04-418Q), 5% human AB serum(Valley Biomedical Inc., #HP1022), 10 mM N-acetyl L-Cysteine(Sigma-Aldrich #A9165) and 100 IU/mL recombinant human IL-2 (NCI/BRBPreclinical Repository). A Jurkat cell line expressing the GreenFluorescent Protein (GFP) upon NFAT activation was maintained inRPMI-1640 medium supplemented with 10% fetal bovine serum (FBS),penicillin and streptomycin. K562 target cells from U. Penn werecultured in IMDM supplemented with 10% FBS.

Effector and Target Cell Engineering with Lentivirus

Pantropic VSV-G pseudotyped lentivirus was produced from Lenti-X 293Tcells (Clontech Laboratories #632180) co-transfected with a pHR′SIN:CSWtransgene expression vector, viral packaging plasmids pCMVdR8.91 andpMD2.G using Lipofectamine LTX (Life Technologies #15338). Infectionmedium supernatant was collected 48 hours after transfection and useddirectly for transduction.

Twenty four hours prior to viral transduction, primary human T cellswere activated using the human T-Activator CD3/CD28 Dynabeads (LifeTechnologies #111-31D) at a 1:3 cell:bead ratio. Jurkat and K562 cellswere split 1-2 days in advance to ensure that cultures would be in logphase at the time of transduction. Transduced Jurkat and K562 cells werecultured for at least 7 days before experiments were conducted. PrimaryT cells were maintained at ˜10̂6/mL in human T cell medium for about twoweeks until cells returned to a resting state. Expression levels of CARsencoded in the lentiviral constructs were quantified by detecting eitherfluorophore-conjugated antibodies or fluorescent reporter proteins usinga flow cytometer.

Quantitation of IL-2 Production and NFAT Activity

Jurkat CD4+ T cells expressing CARs were mixed with cognate ornon-cognate K562 target cells from U. Penn at a 1:2 effector:targetratio. The rapalog A/C Heterodimerizer (Clontech Laboratories #635055)were serially diluted in medium and added to reaction mixtures. After20˜24 hours of incubation, medium supernatants were collected andanalyzed with BD OptEIA Human IL-2 ELISA Set (BD Biosciences #555190).Flow cytometry was performed to quantify NFAT-dependent GFP reporterexpression in Jurkat cells as a separate indicator for CAR activity.

Flow Cytometry-Based Re-Directed Cytotoxicity Assay

The cognate and non-cognate K562 target cells were engineered to expressdistinct fluorescent proteins so that both cell types in a mixture couldbe simultaneously quantified by flow cytometry. The target cell typeswere mixed at a 1:1 ratio and co-incubated with human primary CD8+effector T cells at a 5:2 effector:target ratio. 100 IU/mL human IL-2and varying amounts of the rapalog (Clontech Laboratories #635055) wereadded to reaction mixtures. After 24 hours of incubation, samples werecentrifuged at 400 g for 5 minutes. Pelleted cells were resuspended inwash buffer (PBS+0.5% BSA+0.1% sodium azide) and fixed with an equalvolume of BD Cytofix (BD cat #554655) prior to flow cytometry. Ratios ofthe surviving cognate target cells to non-cognate target cells werecalculated for each sample to enumerate re-directed cytotoxic activitiesof the effector cells.

Results

IL-2 production elicited by the various CAR constructs was assessed. Thedata are presented in FIG. 12.

FIG. 12. IL-2 production triggered by five On-switch CAR variants.Effector=human CD4+ Jurkat T cells engineered with CARs. Target=K562cell lines with or without the cognate CD19 antigen. Amounts of secretedIL-2 by effector cells were quantified by enzyme-linked immunosorbentassay (ELISA).

FIG. 13. IL-2 production by control Jurkat lines in the same experimentas that described in FIG. 12. Construct “125” encodes a conventionalcontrol currently used in clinical trials.

FIG. 14. Comparison between “122+206” and “197+206” in a separateexperiment under conditions identical to those described in FIG. 12.

FIG. 15 demonstrates pharmacologically titratable cytoxicity conferredby the On-switch CAR “197+206” In the presence of the small moleculerapalog, the CAR effectively mediates re-directed cytotoxicity towardscognate target cells. At high dosages of rapalog, this On-switch CAR cansignal as strongly as the “125” conventional CAR. Effector=human primaryCD8+ T cells engineered with CARs or a control vector.Target=fluorescent derivatives of K562 cell lines expressing either thecognate human CD19 antigen or the non-cognate human mesothelin antigen.

FIG. 16 depicts data for CARs constructed with the cytoplasmic tyrosinekinase Zap70 from the T cell receptor pathway as the intracellularsignaling domain.

FIG. 16 shows data from Jurkat cells engineered with several variants ofOn-switch CARs. The engineered Jurkat cells were co-incubated with K562target cells with or without the cognate antigen (CD19) and theindicated concentrations of rapalog. As a CAR component, the Zap70kinase (first and second structures from left featuring “199”) was aseffective as the ITAM (third structure from left featuring “168”) inactivating NFAT function. Addition of the 4-1BB signaling domainincreased surface expression of the antigen recognition portion of thereceptor and led to stronger signaling by “197+199”. A non-signaling CAR(far-right) was included as a negative control.

Example 2: CARs Targeting Mesothelin Materials and Methods

A number of chimeric antigen receptor constructs were made and tested.The constructs shown here encode three different anti-human mesothelinscFv as the antigen recognition domains. FIGS. 19A, 19B, and 19Csummarize the molecular structure of each anti-human mesothelin CAR,with each CAR comprising two polypeptides. The intercellular portion ofeach anti-human mesothelin CAR comprises two 4-1BB co-stimulatorydomains, an FKBP and FRB dimerizer-binding pair, and an ITAMintracellular signaling domain. The three different antigen recognitiondomains shown here are anti-mesothelin HN1 scFv, SS1 scFv, and m912scFv. All membrane-anchored polypeptides are di-sulfide bondedhomo-dimers.

Generation of CAR Constructs

Sequences encoding the anti-mesothelin were cloned from constructs orsynthesized via gene assembly by PCR. The human 4-1BB co-stimulation andCD3 zeta ITAM signaling chains were cloned from cDNAs supplied by OpenBiosystems. HN1 scFv-, SS1 scFv-, and m912 scFv-encoding sequences weresynthesized by PCR and, in some cases, codon optimized. FKBP- andFRB-encoding sequences were cloned from Addgene plasmids.

Standard molecular cloning techniques (polymerase chain reaction (PCR),restriction digestion, ligation, etc.) were applied to generatelentiviral expression plasmids.

Effector and Target Cell Culturing Conditions

A Jurkat cell line expressing GFP upon NFAT activation was maintained inRPMI-1640 medium supplemented with 10% FBS, penicillin and streptomycin.K562 target cells were cultured in IMDM supplemented with 10% fetalbovine serum (FBS).

Effector and Target Cell Engineering with Lentivirus

Pantropic VSV-G pseudotyped lentivirus was produced from Lenti-X 293Tcells (Clontech Laboratories #632180) co-transfected with a pHR′SIN:CSWtransgene expression vector, viral packaging plasmids pCMVdR8.91 andpMD2.G using Lipofectamine LTX (Life Technologies #15338). Infectionmedium supernatant was collected 48 hours after transfection and useddirectly for transduction.

Jurkat and K562 cells were split 1˜2 days in advance to ensure thatcultures would be in log phase at the time of transduction. TransducedJurkat and K562 cells were cultured for at least 7 days beforeexperiments were conducted. Expression levels of CARs encoded in thelentiviral constructs were quantified by detecting eitherfluorophore-conjugated antibodies or fluorescent reporter proteins usinga flow cytometer.

Quantitation of IL-2 Production

Jurkat CD4+ T cells expressing CARs were mixed with cognate ornon-cognate K562 target cells at a 1:2 effector:target ratio. Therapalog A/C Heterodimerizer (Clontech Laboratories #635055) wereserially diluted in medium and added to reaction mixtures. After 20˜24hours of incubation, medium supernatants were collected and analyzedwith BD OptEIA Human IL-2 ELISA Set (BD Biosciences #555190).

Results

IL-2 production elicited by the anti-mesothelin CAR constructs wasassessed. The data are presented in FIG. 19D-F.

FIG. 19. IL-2 production triggered by HN1 scFv (FIG. 19D), SS1 scFv(FIG. 19E), and m912 scFv (FIG. 19F) On-switch CAR variants. IL-2production by a conventional CAR (FIG. 19G, construct #358) was measuredand included for comparison to On-switch CARs (FIG. 19D). Effector=humanCD4+ Jurkat T cells engineered with CARs. Target=K562 cell lines with orwithout the cognate mesothelin antigen. Amounts of secreted IL-2 byeffector cells were quantified by enzyme-linked immunosorbent assay(ELISA).

Example 3: Gibberellic Acid as a Dimerizer of On-Switch CARs Materialsand Methods

FIG. 20A summarizes the molecular structure of the subject gibberellicacid dimerizer CAR. The antigen binding portion comprises the anti-humanCD19 scFv. The intracellular portion comprises two 4-1BB co-stimulatorydomains, a GID1 and GAI dimerizer-binding pair, and an ITAMintracellular signaling domain. All membrane-anchored polypeptides aredi-sulfide bonded homo-dimers.

Generation of CAR Constructs

Sequences encoding the gibberellic acid dimerizer CAR were cloned fromconstructs. The anti-CD19 scFv was cloned from a plasmid. The human4-1BB co-stimulation and CD3 zeta ITAM signaling chains were cloned fromcDNAs supplied by Open Biosystems. GID1- and GAI-encoding sequences werecloned from Addgene plasmids. Standard molecular cloning techniques(polymerase chain reaction (PCR), restriction digestion, ligation, etc.)were applied to generate lentiviral expression plasmids.

Effector and Target Cell Culturing Conditions

A Jurkat cell line expressing GFP upon NFAT activation was maintained inRPMI-1640 medium supplemented with 10% FBS, penicillin and streptomycin.K562 target cells were cultured in IMDM supplemented with 10% fetalbovine serum (FBS).

Effector and Target Cell Engineering with Lentivirus

Pantropic VSV-G pseudotyped lentivirus was produced from Lenti-X 293Tcells (Clontech Laboratories #632180) co-transfected with a pHR′SIN:CSWtransgene expression vector, viral packaging plasmids pCMVdR8.91 andpMD2.G using Lipofectamine LTX (Life Technologies #15338). Infectionmedium supernatant was collected 48 hours after transfection and useddirectly for transduction.

Jurkat and K562 cells were split 1˜2 days in advance to ensure thatcultures would be in log phase at the time of transduction. TransducedJurkat and K562 cells were cultured for at least 7 days beforeexperiments were conducted. Expression levels of CARs encoded in thelentiviral constructs were quantified by detecting eitherfluorophore-conjugated antibodies or fluorescent reporter proteins usinga flow cytometer.

Quantitation of IL-2 Production

Jurkat CD4+ T cells expressing CARs were mixed with cognate ornon-cognate K562 target cells at a 1:2 effector:target ratio. Thegibberellic acid-3 acetoxymethyl ester (gibberrelic acid-3 AM)pre-dissolved in ethanol (Toronto Research Chemicals #G377500) wasdiluted in growth medium and added to reaction mixtures. Gibberellicacid (gibberellic acid-3 AM) was used at 10 mM. After 20˜24 hours ofincubation, medium supernatants were collected and analyzed with BDOptEIA Human IL-2 ELISA Set (BD Biosciences #555190).

Results

IL-2 production elicited by the gibberellic acid dimerizer CAR constructwas assessed. The data are presented in FIG. 20.

FIG. 20. IL-2 production triggered by gibberellic acid dimerizer CARvariant (FIG. 20B). IL-2 production by a conventional CAR (FIG. 20C,construct “125”) was measured and included for comparison to On-switchCAR. Effector=human CD4+ Jurkat T cells engineered with CARs.Target=K562 cell lines with or without the cognate CD19 antigen. Amountsof secreted IL-2 by effector cells were quantified by enzyme-linkedimmunosorbent assay (ELISA).

Example 4: On-Switch CARs with Various Co-Stimulatory Domains Materialsand Methods

A number of chimeric antigen receptor constructs were made essentiallyas described for Example 1, except various other co-stimulatory domainswere exchanged for the 4-1BB co-stimulatory domains. FIGS. 21A and 21Bsummarize the molecular structure of the CARs described here.

Generation of CAR Constructs

Sequences encoding the anti-human CD19 scFv were cloned from a plasmid.The human CD3 zeta ITAM signaling chain and the human co-stimulatorydomains CD28 and OX-40 encoding sequences were cloned from cDNAssupplied by Open Biosystems. FKBP- and FRB-encoding sequences werecloned from plasmids from Addgene.

Standard molecular cloning techniques (polymerase chain reaction (PCR),restriction digestion, ligation, etc.) were applied to generatelentiviral expression plasmids.

Testing of CAR Constructs

Effector and target cells are cultured and transfected according toExample 1 using the on-switch CAR CD28 and OX-40 co-stimulatory domaincontaining constructs described (FIG. 21A-B, constructs “365+367” and“399+400”, respectively) and corresponding conventional CAR controls(FIG. 21C-D, constructs “366” and “398”, respectively). IL-2 production,NFAT activity assays, and flow cytometry-based assays can also beperformed with the CD28 co-stimulatory domain containing construct andOX-40 co-stimulatory domain containing construct as described forExample 1. Alternatively, subunits of on-switch CAR CD28 and OX-40co-stimulatory domain containing constructs can be paired with subunitsof constructs from Example 1 (e.g., “197+367”, “365+206,” “197+400”,“399+206,” etc.).

Example 5: In Vivo Assessment of On-Switch CAR

An On-switch CAR can be assessed for its ability to mediate in vivokilling of a target tumor cell. In vivo tumor cell killing elicited byinjection of T cells expressing the ON-switch CAR is assessed. Tumorcell lines that have been confirmed in vitro to express the cognateantigen and can be killed by CD8⁺ T cells expressing the correspondingCAR are used. Tumor cells engineered to express either the firefly orRenilla luciferase to enable bio-luminescence imaging to quantify tumorburden in vivo can be used. Tumor cells are injected intoimmunocompromised mice (e.g., 6˜10 week old female NOD scid gamma (NSG)mice) either subcutaneously for subcutaneous tumor models orintravenously for systemic tumor models. The method of tumorimplantation and the optimal number of tumor cells to implant can bebased on conditions optimal for the tumor cell line used. Tumor burdencan be monitored twice a week by bio-luminescence imaging and by calipermeasurement when applicable. As soon as tumor burden is detectable,0.5˜2.5×10̂7 total T cells (1:1 CD4⁺:CD8⁺) expressing the ON-switch CARare intravenously injected into mice to begin treatment. A dimerizingsmall molecule drug (e.g., rapalog) is administered intraperitoneally ina vehicle formulation. On-switch CAR-expressing T cells can be injectedrepeatedly during the experiment to enhance the anti-tumor effect.Interleukin-2 (IL-2) can be administered to enhance the anti-tumoreffect.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1.-36. (canceled)
 37. A method of treating a cancer in an individual,the method comprising: A) introducing a T lymphocyte into theindividual, wherein the T lymphocyte is genetically modified to producea heterodimeric chimeric antigen receptor (CAR) comprising a firstpolypeptide and a second polypeptide, wherein: a) the first polypeptidecomprises: i) an extracellular antigen binding domain; ii) atransmembrane domain; and iii) a first member of a dimerization pair;and b) the second polypeptide comprises: i) a transmembrane domain; ii)a second member of the dimerization pair; and iii) an intracellularsignaling domain comprising an immunoreceptor tyrosine-based activationmotif (ITAM), wherein the intracellular signaling domain provides signaltransduction activity, wherein the first polypeptide, the secondpolypeptide or both the first and second polypeptides comprise anintracellular costimulatory polypeptide, and wherein the antigen-bindingdomain of the heterodimeric, conditionally active CAR is specific for anepitope on a cancer cell in the individual; and B) administering to theindividual an effective amount of a dimerizing agent, wherein thedimerizing agent induces dimerization of the heterodimeric,conditionally active CAR, wherein said dimerization provides foractivation of the T lymphocyte, thereby treating the cancer.
 38. Themethod of claim 37, wherein the first polypeptide comprises a hingeregion interposed between the extracellular antigen binding domain andthe transmembrane domain.
 39. The method of claim 38, wherein the hingeregion is an immunoglobulin IgG hinge region or a hinge derived fromCD8.
 40. The method of claim 37, wherein the intracellular signalingdomain comprising the ITAM is selected from the group consisting ofCD3-zeta and ZAP70.
 41. The method of claim 37, wherein the first andsecond members of the dimerization pair are selected from the groupconsisting of: a) FK506 binding protein (FKBP) and FKBP-rapamycinassociated protein (FRB); b) a Gibberellic Acid Insensitive (GAI)protein and a gibberellin receptor (GID1) protein; c) FKBP andcalcineurin catalytic subunit A (CnA); d) an abscisic acid receptor(PYL) protein and an abscissic acid insensitive (ABI) protein; e) acryptochrome 2 (Cry2) protein and a transcription factor bHLH63 (CIB1)protein; and f) FKBP and cyclophilin.
 42. The method of claim 37,wherein the first and second members of the dimerization pair areselected from the group consisting of: a) FK506 binding protein (FKBP)and FKBP; b) gyrase B (GyrB) and GyrB; c) dihydrofolate reductase (DHFR)and DHFR; and d) DmrB and DmrB;
 43. The method of claim 37, wherein thefirst member of the dimerization pair is an FK506 binding protein(FKBP), and wherein the second member of the dimerization pair is aFKBP-rapamycin associated protein (FRB).
 44. The method of claim 43,wherein the FKBP comprises an amino acid sequence having at least 95%amino acid sequence identity to the amino acid sequence set forth in SEQID NO:12, and wherein the FRB comprises an amino acid sequence having atleast 95% amino acid sequence identity to the amino acid sequence setforth in SEQ ID NO:14.
 45. The method of claim 37, wherein the firstmember of the dimerization pair is GID1, and wherein the second memberof the dimerization pair is GAI.
 46. The method of claim 45, wherein theGID1 polypeptide comprises an amino acid sequence having at least 95%amino acid sequence identity to the amino acid sequence set forth in oneof SEQ ID NOs:95-97, and wherein the GAI polypeptide comprises an aminoacid sequence having at least 95% amino acid sequence identity to theamino acid sequence set forth in SEQ ID NO:94.
 47. The method of claim37, wherein the cancer cell is a breast cancer cell, a B cell lymphoma,a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell,a mesothelioma, a lung cancer cell, a non-Hodgkin B-cell lymphoma cell,an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, amelanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocyticleukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, amedulloblastoma, or a colorectal cancer cell.
 48. The method of claim37, wherein the intracellular costimulatory polypeptide is selected fromthe group consisting of: 4-1BB, CD28, ICOS, OX-40, BTLA, CD27, CD30,GITR, and HVEM.
 49. The method of claim 48, wherein the 4-1BBpolypeptide comprises an amino acid sequence having at least 95% aminoacid sequence identity to the amino acid sequence set forth in SEQ IDNO:24.
 50. The method of claim 48, wherein the OX-40 polypeptidecomprises an amino acid sequence having at least 95% amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO:65.
 51. Themethod of claim 48, wherein the CD28 polypeptide comprises an amino acidsequence having at least 95% amino acid sequence identity to the aminoacid sequence set forth in SEQ ID NO:63.
 52. The method of claim 37,wherein the first polypeptide comprises a first costimulatorypolypeptide comprising an amino acid sequence and the second polypeptidecomprises a second costimulatory polypeptide comprising an amino acidsequence that has at least 95% amino acid identity to the amino acidsequence of the first costimulatory polypeptide in the firstpolypeptide.
 53. The method of claim 37, wherein the intracellularsignaling domain comprising the ITAM comprises an amino acid sequencehaving at least 95% amino acid sequence identity to the amino acidsequence set forth in one of SEQ ID NOs:98-119.
 54. The method of claim37, further comprising administering to the individual a cancerchemotherapeutic agent, an antibody, or a biological response modifier.55. The method of claim 37, wherein the first member and the secondmember of the dimerization pair are extracellular.
 56. The method ofclaim 37, wherein the first member and the second member of thedimerization pair are intracellular.